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MITOSIS-SPECIFIC PHOSPHORYLATION OF MAP4 AND CHARACTERIZATION OF THE MPM-2 EPITOPE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Yunhi Choi, B.S, M.S ★ ★ ★ ★ ★
The Ohio State University 1996
Dissertation Committee: Approved by Dale D.Vandre, Ph.D. John M.Robinson, Ph.D. Arthur R.Strauch, Ph.D. .dviser R.Tom Boyd, Ph.D. Ohio State Biochemistry Program UMI Number: 9620003
UMI Microform 9620003 Copyright 1996, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 TO MY PARENTS
ii ACKNOWLEDGMENTS
I wish to express my sincere appreciation to my adviser, Dr. Dale D.Vandre, for his guidance, support, and insight throughout my graduate study. I also thank him for his patience, understanding, and encouragement.
I would like to thank to members of my dissertation committee, Drs. John M.Robinson, Arthur R.Strauch, and R.Tom Boyd for their time, helpful suggestions, and interest in my studies.
Special thanks to my colleagues, Min Ding, Yang Feng, Christine Kondratick, and Colin Lowry, for their friendship, encouragement, sharing their ideas, and support.
I extend my gratitude to my family, especially my parents and my mother-in-law for thier love and support. Finally, I wish to thank to my wonderful husband, Siyoung, and my precious son, Jaesuk, for their love and patience in allowing me to complete my studies.
iii VITA
September 26, 1965 ------Born - Seoul, Korea 1988 ------B.S. Department of Biochemistry Yonsei University Seoul, Korea 1990 ------M.S. Department of Biochemistry Yonsei University Seoul, Korea 1991-1995 ------Graduate Research Associate Ohio State Biochemistry program, The Ohio State University, Columbus, Ohio
PUBLICATIONS
Choi, Y., Wills,V.L., and Vandre, D.D.(1993) Mitosis dependent phosphorylation of MAP4 and 125 kD MAP. Mol. Biol. Cell 4: 269a. [Abstract] Choi, Y. and Vandre, D.D.(1994) Interphase MAP4 becomes MPM-2 immunoreactive following phosphorylation by cdc2 kinase. M o l .Biol. Cell 5: 168a. [Abstract]
Choi, Y. and Vandre, D.D.(1995) Phosphorylation of interphase MAP4 and formation of the mitosis-specific MPM-2 phosphoepitope in vitro. J . Biol. Chem. [manuscript submitted]
FIELD OF STUDY
Major field: Biochemistry
v TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii VITA ...... iv LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF ABBREVIATIONS ...... x ABSTRACT ...... xi CHAPTERS I. INTRODUCTION ...... 1 II. MATERIALS AND METHOODS ...... 19 III. RESULTS ...... 31 IV. DISSCUSSION ...... 102 LIST OF REFERENCES ...... 116
vi LIST OF TABLES
TABLES PAGE 1. Purification of microtubules and MAP4 from HeLa cells ...... 50
2. Potential phosphorylation sites are present on and conserved betweendifferent MAP4s ...... 51
3. Fractionation of microtubule-associated kinases from mitotic HeLa cells ...... 52
vii LIST OF FIGURES
FIGURES PAGE 1. Purification and immunoblot analysis of MAP4 and 125 kD MAP from interphase and mitotic HeLa cells ...... 55
2. Cell cycle dependent phosphorylation of HeLa MAP4 ...... 57
3. In vitro phosphorylation of MAP4 by exogenous kinases ...... 59
4. Assay of endogenous kinase activity and analysis of proteins associated with mitotic HeLa microtubules ...... 61
5. Phosphorylation of interphase HeLa MAP4 by endogenous kinases present in mitotic HeLa samples ...... 63
6. Endogenous kinase activities of mitotic HeLa samples under different buffer conditions ...... 65
7. Effects of MAP4 substrate concentration on the HeLa microtubule-associated kinase activity ...... 67
8. Time-course of interphase HeLa MAP4 phosphorylation by p34cdc2 kinase ...... 69
9. Phosphorylation of interphase HeLa MAP4 by mitotic HeLa microtubule-associated kinase activities ...... 71
10. Phosphorylation of interphase HeLa MAP4 by kinase activities present in mitotic HeLa microtubule salt-extracted supernatant or salt-extracted pellet ...... 73
viii FIGURES PAGE 11. Phosphorylation of interphase HeLa MAP4 by mitotic HeLa microtubule-depleted supernatant ..75
12. The effect of phosphatase inhibitors on the phosphorylation of interphase HeLa MAP4 by mitotic microtubule-depleted supernatant ...... 77
13. Fractionation of mitotic HeLa microtubule-depleted supernatant kinase activities by ammonium sulfate ...... 79
14. Analysis of distribution of human Plk 1 in mitotic HeLa cells ...... 81
15. Immunoprecipitation of Plk 1 from mitotic HeLa cell ...... 83
16. In vitro kinase assay of Plk 1 immunoprecipitate ...... 85
17. Time-course of phosphorylation of interphase HeLa MAP4 by Plk 1 immunoprecipitate ...... 87
18. Immunoprecipitation of Plk 1 and MAP4 indicate they are not associated ...... 89
19. Peptide mapping of mitotic HeLa MAP4 by NCS cleavage ...... 91
20. Peptide mapping of mitotic HeLa MAP4 by trypsin digestion ...... 93
21. Peptide mapping of mitotic HeLa MAP4 by chymotrypsin digestion ...... 95
22. Peptide mapping of mitotic HeLa MAP4 by Staphylococcus aureus V8 protease ...... 97
23. Microtubule binding assay of MAP4 peptide fragments generated by V8 protease digestion ...... 99
24. Peptide mapping of mitotic HeLa MAP4 by endoproteinase Arg-C ...... 101
ix LIST OF ABBREVIATIONS
AEBSF 4-(2-Aminoethyl)-benzenesulfonyl fluoride APMSF Amidinophelylmethylsulfonyl fluoride CAK Cyclin dependent kinase activating kinase CDK Cyclin dependent kinase ERK Extracellular regulated kinase
kD kiloDaltons MAP Microtubule-associated protein MAPK Mitogen-activated protein kinase MPF Maturation promoting factor MPM-2 Mitotic protein monoclonal antibody-2 NCS N-chlorosuccinimide PBS Phosphate buffered saline PKC Protein kinase C Plk 1 Polo-like kinase 1 PMSF Phenylmethylsulfonyl fluoride PVDF Polyvinylidine difluoride SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TBS Tris buffered saline
x ABSTRACT
Microtubule-associated proteins (MAPs) bind to microtubules and regulate their assembly in vitro. Phosphorylation of MAPs appears to regulate their affinity for microtubules, and changes in the phosphorylation state of MAPs may provide a mechanism for regulating microtubule dynamics in vivo. In particular, MAP phosphorylation may play an important role in microtubule rearrangements associated with the transition from interphase to mitosis. MAP4 is the major MAP found in non-neuronal tissues, and HeLa cell MAP4 is demonstrated to be phosphorylated in a cell cycle dependent fashion. Mitotic MAP4 is also reactive with the mitotic phosphoepitope specific monoclonal antibody MPM-2. It is shown here that interphase and mitotic HeLa
cell MAP4 are both substrates for MAP kinase, p34cdc2
kinase, and polo-like kinase (plk 1) but they are not substrates for casein kinase-2 in vitro. Further, extensive in vitro phosphorylation of interphase
HeLa MAP4 by p34cdc2 kinase converts it into an MPM-2 reactive form. Endogenous mitotic HeLa cell
xi microtubule-associated kinase activity has been identified that will also convert interphase HeLa MAP4 into an MPM-2 reactive protein. In comparison to the p34cdc2 kinase, the endogenous HeLa microtubule-associated kinases are much more efficient at forming the MPM-2 epitope on MAP4. The characteristics of these endogenous microtubule-associated kinases suggest they are similar to p34cdc2 kinase. Therefore, the MAP4 MPM-2 epitope kinase appears to be microtubule-associated and related to cyclin-dependent kinases. The MPM-2 epitope on HeLa cell MAP4 was shown to be located in the proline-rich region of the protein, which contains conserved consensus phosphorylation sites for proline-directed kinases. This result is consistent with the possibility that the MPM-2 epitope kinase of MAP4 is a cdc2-like kinase. CHAPTER I
INTRODUCTION
Cellular growth and division are fundamentals of biology. Most cells grow, replicate their DNA, segregate their chromosomes into daughter cells, and divide during the cell cycle. The cell cycle can be divided into two essential parts: interphase and mitosis. During interphase, DNA replication and synthesis of cellular proteins needed for growth occur. Mitosis begins with a period called prophase, during which chromosome condensation and nuclear envelope breakdown occur. Following nuclear envelope breakdown, the cytoplasmic microtubule network disassembles, forming the mitotic spindle. Each pair of sister chromatids are attached to the spindle poles via the kinetochore microtubules and lined up midway between the two spindle poles during metaphase. The cell remains for a short time in metaphase before the transition from mitosis back to interphase, which begins with the onset of anaphase. Anaphase onset marks the trigger of the exit from mitosis, and is
1 initially characterized by the splitting of the sister chromatids. During the exit from mitosis, chromosome decondensation, nuclear envelope reformation, and reestablishment of interphase microtubule array occur.
Mitosis is a relatively short period representing about 5% of the total cell cycle. However, dramatic changes in the cytoarchitecture of the cell takes place during this period. The microtubule array is reorganized to form the mitotic spindle which is involved in the segregation of the replicated chromosomes into the two daughter cells. Thus, the control of assembly and disassembly of the mitotic apparatus is essential for the regulation of cellular proliferation.
Although the mechanism that regulates the transition from interphase to mitosis has not been completely characterized, progression of eukaryotic cells into mitosis involves a dramatic increase in the level of protein phosphorylation (Capony et al., 1986; Lohka et al., 1987), and mitosis specific phosphorylation of certain proteins appears to be an important regulatory process. For example, phosphorylation of the nuclear lamina proteins has been reported to be involved in nuclear envelope breakdown (Dessev et al., 1989), and phosphorylation of histones is thought be involved in chromosome condensation during mitosis (Langan et al., 1989) . Phosphorylation of vimentin has been shown to 3 induce intermediate filament reorganization during mitosis (Chou et al., 1990). These mitosis specific phosphorylations are correlated directly, or indirectly with the kinase activity called maturation promoting factor (MPF), which has been considered as a universal trigger of the G2 to M phase transition (Nurse, 1990; Pines, 1995).
MPF, also called M-phase kinase, is composed of two components, a polypeptide of relative molecular mass between 45,000-62,000 (Mr, 45-62 kD) known as mitotic cyclins, and a 34 kD catalytic subunit which has serine/threonine kinase activity known as p34cdc2 or cdc2 kinase (Moreno and Nurse, 1990). Mitotic cyclins are proteins that accumulate continuously during interphase and are then quantitatively degraded at the metaphase- anaphase transition in mitosis.
Two general classes of mitotic cyclins have been found in virtually all eukaryotes and are termed A- and B- type cyclins. They can be distinguished by distinct sequence motifs and by their kinetics of accumulation and degradation (Minshull et al., 1990). A-type cyclins are synthesized earlier in the cell cycle than B-type cyclins and are degraded prior to entry into M phase. In most organisms, there are two closely related B-type cyclins, termed Bl and B2. Activation of cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins. Cyclin binding leads to phosphorylation of cdc2 on threonine-161 by the cyclin-dependent kinase (CDK)-activating kinase (CAK). This phosphorylation makes cdc2 kinase active, however, concomitant inhibitory phosphorylations of cdc2 on threonine-14 and tyrosine-15 suppress the activity of MPF during interphase (Draetta, 1993; Pines 1993; Solomon et al., 1990), and this complex is called "pre-MPF". The weel protein kinase has been shown to phosphorylate cdc2 on tyrosine-15 but not threonine-14 (McGowan and Russell, 1993; Parker and Pinwica-Worms, 1992), and recently another cdc2-inhibitory kinase called mytl was identified from Xenopus (Mueller et al., 1995b). Mytl is a member of the weel kinase family, but unlike weel, mytl phosphorylates both threonine-14 and tyrosine-15 and it is associated with plasma membranes (Mueller et al., 1995b). These cdc2-inhibitory kinases, weel and mytl, are highly regulated during the cell cycle, activities being high during interphase but low at mitosis as a result of extensive phosphorylation (Mueller et al., 1995a; Mueller et al., 1995b). At the onset of mitosis, activation of the "pre-MPF" occurs by dephosphorylation of threonine-14 and tyrosine-15 residues which is mediated by the phosphatase called cdc25 (Dunphy and Kumagai, 1991; Gautier et al., 1991). The cdc25 activity has also been shown to be regulated during the cell cycle; the 5 phosphatase activity is low during interphase and increased in M-phase by phosphorylation (Izumi et al., 1992; Kumagai and Dunphy, 1992). Threonine-14 and tyrosine-15 are located within the ATP binding site of cdc2 and must be dephosphorylated in order to activate the kinase. Finally, cyclin destruction leads to inactivation of MPF. The activity of MPF peaks during metaphase and drops rapidly as cells exit from M-phase.
Although the activation mechanism of cdc2 kinase is known in detail, the mechanism by which MPF regulates M-phase events is largely unknown. Several potential substrates for cdc2 kinase have been reported (Moreno and Nurse, 1990), however, the identification of in vivo substrates needs to be carried out for complete understanding of the mechanism of mitotic control by MPF.
The cdc2 gene product is known to control cell cycle progression in yeast at the Gl/S and G2/M transition (Norbury and Nurse, 1992). In animal cells, homologs of the yeast cdc2 gene are essential for the initiation of the M phase (Norbury and Nurse, 1992), while the Gl/S transition requires cdk2, a cdc2-related protein kinase (Tsai et al., 1991; Elieage et al., 1992). Cdc2 is thus the first identified member of kinases, the cyclin- dependent protein kinases (cdks), whose activity is dependent upon the association of specific regulatory subunits known as cyclins (Meyerson et al., 1992). 6 It is known that cdc2 kinase- activity requires cyclin B (Draetta, 1990). In addition to the B-type cyclins, a family of structurally related proteins including cyclins A, C, D (Dl, D2, and D 3 ) and E have been identified (Wang et a l ., 1990; Lew et a l ., 1991; Xiong et al., 1991; Motukura et al, 1991; Koff et al., 1991). Complex formation between cdk proteins and cyclins appears to be regulated during the different stages of the cell cycle. The presence of a family of cdc2-related kinases suggests that they may have distinct functions in the regulation of the cell cycle in response to different signals. Since the regulation of cell cycle progression may be more complex in a multicellular organism in comparison to a unicellular organism, a specific cdc2-related protein may have specialized functions at different points in the cell cycle.
Eukaryotic cells contain an elaborate cytoskeleton, a complex network of protein filaments that extends throughout the cytoplasm. The cytoskeleton is responsible for a variety of cellular functions in addition to its function in forming the internal framework of the cell. The cytoskeleton regulates various types of movement that take place within eukaryotic cells. For example, muscle contraction, cilia and flagella beating, mitotic movements of chromosomes, and organelle and vesicle transport are all controlled by cytoskeletal components. The cytoskeleton is also involved in morphological and 7 structural changes that occur during cellular proliferation and differentiation. These activities of the cytoskeleton are carried out by three principal types of protein filaments : actin filaments, microtubules, and intermediate filaments.
Microtubules are heterodimers of a- and ^-tubulin, assembled through self-association. The tubulin dimers, building blocks of microtubules, are arranged in a polarized manner within the microtubule lattice, giving a structural polarity to the microtubule. The assembly of microtubules are nucleated by the centrosome complex in vivo, with the plus ends of the microtubules distal to the 4 centrosome. The centrosomal components which are responsible for the nucleation of microtubules are not completely understood, but it has been suggested that y- tubulin might be responsible for the polarized nucleation of microtubules (Oakley et al., 1990; Joshi et al., 1992).
Microtubules are highly dynamic structures and undergo dramatic changes during the transition from interphase to mitosis. During interphase, microtubules are arrayed in a radial network emanating from the perinuclear centrosome. At the onset of mitosis, the interphase microtubule network is destroyed and microtubules reorganize into the mitotic spindle. Microtubules also form a number of specialized arrays in different cell types including 8 parallel array oriented from the apical to basal surface in polarized epithelial cells, and staggered linear arrays in axons. The ability of cells to from various arrays of microtubules is due to the intrinsic dynamic instability of microtubules. The molecular mechanism regulating microtubule dynamics in vivo are not completely understood. However, the microtubule-associated proteins or MAPs have been identified as a possible regulatory factor of microtubule dynamics in vitro.
Microtubule-associated proteins (MAPs) consist of a group of proteins that bind to microtubules in vivo and copurify with microtubules in vitro following temperature induced cycles of polymerization. The most abundant and well characterized MAPs have been identified in mammalian brain (Cleveland et al., 1977; Kim et al., 1979), and they have been grouped into three classes designated MAPI (300-350 kD) , MAP2 (270 kD), and the tau group with a molecular weight about 60 kD (Sloboda et al., 1975). MAPI and MAP2 have several isoforms as determined by electrophoretic mobility (Bloom et al., 1984). Thus, these high molecular weight neuronal MAPs consist of multiple polypeptides termed MAPIA, MAPIB, MAP1C, MAP2A, MAP2B, and MAP2C (Bloom et al., 1984). These neuronal MAPs have been shown to induce tubulin polymerization in vitro and bind to the reconstituted microtubules. These MAPs are expressed almost exclusively in neuronal tissues (Olmsted et al., 1986) . Their restricted distribution implies that these proteins have specialized functions in the nervous system such as construction of specialized cytoskeleton'in neurites (Chapin and Bulinski, 1991), however, their in vivo functions are essentially unknown. Similarly, discrete MAPs present in non-neuronal tissues and cultured cells may also have specialized functions, as well as roles that are associated with cell division and the formation and function of the mitotic spindle.
The most abundant high molecular weight MAPs present in non-neuronal cells include a family of proteins designated MAP4. The MAP4 proteins have an apparent molecular mass of about 200 kD on SDS gels, and are distributed ubiquitously among mammalian tissues and cells. The ubiquitous distribution of MAP4 implies that this MAP may contribute to fundamental cellular processes which depend on microtubules. Members of the MAP4 family include the HeLa 210 kD MAP (Chapin and Bulinski, 1991; West et al., 1991), mouse MAP4 (West et al., 1991), and bovine 190 kD MAP (MAP-U) (Aizawa et al., 1990). These MAPs are thermostable and exist as complexes of related polypeptides (West et al., 1991). The full length coding sequences of MAP4 derived from human, mouse, and bovine MAP4 (West et al., 1991; Aizawa et al., 1990) have been identified, and derived structural features of the protein have been examined. MAP4 is encoded by a single gene expressing multiple mRNAs (West et al., 1991; Aizawa et al., 1991). Amino acid sequence analysis have shown that 10 human, mouse, and bovine MAP4 share nearly 75% sequence homology.
Structurally, MAP4 consists of an acidic projection domain that is located in the N-terminal region, a basic microtubule binding domain in the C-terminal region, and a short acidic tail (West et al., 1991; Aizawa et al., 1990; Aizawa et al., 1991). The microtubule binding domain of MAP4 is homologous to that of MAP2 and smaller neuronal tau MAPs (Chapin et al., 1991). A conserved repeated 18 amino acid motif separated by less highly conserved spacer sequences of 13-14 amino acids comprises a tubulin-binding domain common to this group of MAPs (Lee, 1990). The N- terminal projection domain of these MAPs, which extends away from the microtubule lattice, are considered to be involved in the formation of microtubule cross-bridges and the association of microtubules with other cytoskeletal elements (Sherline et al., 1977). The N-terminal domain of MAP4 shows no significant homology with neuronal MAPs, including MAP2 and tau, suggesting that this domain may have specific functions for individual MAPs such as interactions with other intracellular components. Located between the N-terminal projection domain and the C- terminal microtubule-binding domain of MAP4, there is a region rich in proline. This proline-rich region is shown to enhance the binding of the tubulin binding domain to microtubules in vitro (Aizawa et al., 1991). Recently, an additional HeLa cell MAP, the 125 kD MAP designated 11 ensconsin, has been identified and partially characterized (Bulinski'and Bossier, 1994).
MAP4 promotes the assembly of microtubules in vitro (Aizawa et al., 1991; Murofushi et al., 1986; Hoshi et al., 1992), but the functions of this protein in vivo are not fully delineated. Immunological data have shown that MAP4 is associated with interphase microtubules and mitotic spindle fibers (Aizawa et al., 1991; Izant et al., 1982; Izant et a l ., 1983; Olmsted et al., 1986), suggesting that MAP4 may play an important role in the formation and destruction of these microtubular structures in a cell-cycle dependent manner. As mentioned above, the interphase microtubule network is destroyed and mitotic spindle is formed during the G2 to M phase transition. The mitotic spindle microtubules are more dynamic than interphase microtubules (Gorbsky and Borisy, 1989). It has been reported that the formation and dynamic stability of microtubules are regulated by binding of MAPs (Drubin
T and Kirchner, 1986; Lewis et al., 1989). Phosphorylation of MAPs has been suggested as a mechanism for modulating binding of MAPs to microtubules, thereby controlling microtubule dynamic stability. Phosphorylation of major neuronal MAPs, such as MAP2 and tau by protein kinase C (PKC) has been shown to reduce the activity of these MAPs to promote microtubule assembly (Hoshi et al., 1988; Hoshi et al., 1987). PKC or cdc2 kinase phosphorylation of the proline-rich region of bovine MAP4 suppresses the activity 12 of MAP4 to induce microtubule assembly (Aizawa et al., 1991; Mori et al., 1991). Hoshi et al (1992) reported that phosphorylation of MAP2 and MAP4 by MAP kinase resulted in reduced ability of these MAPs to induce tubulin polymerization. The above reports indicate that the functional properties of specific MAPs might be regulated by altering the phosphorylation state of the protein.
Since MAP4 is distributed ubiquitously in many types of cells and is the predominant MAP in non-neuronal cells, phosphorylation of MAP4 in a cell cycle dependent manner may contribute to the changes in microtubule dynamics and the structural rearrangement of microtubules that occur during mitosis (Verde et al., 1992; Ookata et al., 1993).
Recently, p34cdc2/cyclin B complex has been shown to be associated with microtubules through binding between cyclin B and MAP4 (Ookata et al., 1995). Ookata et al (1995) demonstrated that cyclin B binds to the proline- rich region of MAP4, and phosphorylation of MAP4 by p34cdc2/cyclin B diminished the microtubule stabilizing activity of MAP4. Thus, their data suggests that the interaction of MAP4 with p34cdc2/cyclin B may be important for the regulation of microtubule dynamics during mitosis by targeting the M-phase kinase to appropriate substrates. 13 MAP4 phosphorylated during mitosis reacts with the MPM-2 antibody (Vandre et al., 1991). The MPM-2 monoclonal antibody was originally raised against extracts of mitotic HeLa cells and selected by preferential staining of mitotic versus interphase cells (Davis et al., 1983). It has been shown that this antibody recognizes a group of phosphoproteins that contain related phosphoepitopes during M-phase. The localization of proteins containing the MPM-2 epitope on key structural elements in M-phase, such as spindle poles, kinetochores, the midbody, and chromosomes (Vandre et al., 1984; Engle et a l ., 1988; Hirano and Mitchison, 1991), suggested that phosphorylation of the MPM-2 epitope might be involved in the regulation of microtubule dynamics, spindle assembly, and spindle function. Furthermore, the phosphorylation of the MPM-2 epitope during the G2 to M transition is a highly conserved phenomenon, indicating that it could be a regulatory mechanism by which several mitotic events are regulated. Treatment of mitotic centrosomes with MPM-2 has been shown to inhibit nucleation of microtubules (Centonze et al., 1990). Microinjection of the MPM-2 antibody into Xenopus oocytes prior to progesterone stimulation also resulted in an inhibition in the appearance of MPF activity (Kuang et al., 1989). In addition, MPM-2 antibody microinjected into Xenopus M- phase eggs resulted in diminished MPF activity, suggesting that the MPM-2 antibody may bind to MPF or a protein regulating MPF activity (Kuang et al., 1989). 14 Since MPF itself does not react with the MPM-2 antibody (Kuang et al., 1994), proteins that regulate MPF activity should contain the MPM-2 phosphoepitopes (Taagepera et al., 1994).
The significance of the MPM-2 phosphoepitope in mitotic progression has been addressed in many different organisms. For example, the MPM-2 antibody has been used to investigate the mechanism of sperm aster formation in rabbit zygotes (Pinto-Correia et al., 1994). The sperm midpiece, which is responsible for sperm aster formation, contains antigens recognized by the MPM-2 antibody. These MPM-2 reactive phosphoproteins were demonstrated to be essential components in fertilization of the rabbit oocyte (Pinto-Correia et al., 1994). Pretreatment of the sperm midpiece with MPM-2 blocked aster formation in oocytes activated by calcium stimulation, indicating that the activity of MPM-2 antigens was necessary for aster formation (Pinto-Correia et al., 1994). Their studies also showed that onset of the loss of MPM-2 reactivity coincided with aster formation, which marks the initiation of anaphase. MPM-2 phosphoepitopes are thought to be involved in the nucleation of microtubules by centrosomes and dephosphorylation of MPM-2 epitopes has also been associated with anaphase onset in somatic cells (Centonze and Borisy, 1990; Vandre and Borisy, 1989). MPM-2 reactive phosphoproteins have been shown to be involved in the progression of cell cycle in plant cells as well. 15 The localization of MPM-2 immunoreactive materials was examined in synchronous populations of Vicia faba root meristem cells and isolated nuclei (Binarova et al. , 1993). The presence of MPM-2 reactive phosphoepitopes not only indicate phylogenetic conservation, but the MPM-2 reactive phosphoproteins associated with Vicia faba kinetochores imply that common mechanisms are involved in the regulation of mitosis in both plants and animals (Binarova et al., 1993). Engle et al (1988) showed that MPM-2 staining colocalized with the spindle pole body of Aspergillus nidulans and this staining varied during the cell cycle.
Recently, cdc25, a positive regulator of MPF, and weel and mytl, both negative regulators of MPF, have been found to be MPM-2 antigens (Kuang et al., 1994; Mueller et al., 1995a; Mueller et al., 1995b). The NIMA protein kinase, which has been reported to be essential for mitotic initiation in Aspergillus nidulans, has also been found to be an MPM-2 antigen (Ye et al., 1995). NIMA is activated during G2 phase by hyperphosphorylation and becomes MPM-2 reactive. Phosphorylation of NIMA by p34cdc2/cyclin B in vitro generated an MPM-2 epitope on NIMA (Ye et al., 1995) . Other proteins known to be MPM-2 reactive include MAPlB (Tombes et al., 1991; Vandre et al., 1986), DNA topoisomerase II a and II P present in mitotic chromosomes
(Taagepera et al., 1993), and activated p42mapk (Taagepera 16 et al., 1994). The identification of MPM-2 antigens as important cell cycle regulatory proteins indicates the possible significance of the MPM-2 epitope and the MPM-2 epitope kinase(s) (ME kinase(s)) responsible for generating those sites.
The MPM-2 antibody recognizes mitotic phosphoproteins in every mammalian species examined (Vandre et al., 1986), and mitotic MAP4 in both CHO and HeLa cells (Vandre et al., 1991). Therefore, the MPM-2 epitope on MAP4 is expected to be conserved among mammalian species. The long-term goal of our work is to identify the phosphorylation sites on MAP4 that are recognized by the MPM-2 antibody and to determine the kinase(s) which phosphorylate the MPM-2 epitope.
The amino acid sequence analysis of MAP4 shows several consensus sites for a variety of different protein kinases. Specifically, there are 21 serine or threonine residues in human MAP4 that are followed by a proline residue, providing several potential sites for proline- directed serine/threonine kinases. Comparison of the MAP4 sequence from human, mouse, and bovine revealed that several consensus motifs for p34cdc2, MAP kinase, and casein kinase-2 are common to these species. Two sites
for p34cdc2 and three casein kinase-2 sites are conserved in all MAP4 sequences available to date. Since the above 17 kinases have been indicated in the regulation of the cell cycle (Mori et al., 1991; Aizawa et al., 1991; Hoshi et al., 1992; Tombes et a l ., 1991), conservation of these phosphorylation sites on MAP4 might suggest that these kinases are candidate MPM-2 epitope kinases, and that the conserved sites are potential MPM-2 epitope sites.
Despite the presence of known consensus phosphorylation sites on MAP4, it cannot be excluded that an unidentified novel protein kinase may be responsible for the phosphorylation of the MPM-2 epitope. For example, human polo-like kinase (Plk 1) is a novel protein kinase family implicated in cell cycle progression (Golsteyn et al., 1995). Plk 1 is a homolog of polo, a serine/threonine- specific kinase first identified in Drosophila, which has been implicated in controlling spindle function and chromosome segregation (Llamazares et al., 1991). Saccharomyces cerevisiae cdc5p and human plk 1 have been reported to be functional homologs of Drosophila polo (Kitada et al., 1993; Golsteyn et al., 1994). Human Plk 1 has been reported to be cell cycle regulated, activity being low during interphase but high during mitosis. Plk 1 has also been shown to associate with the spindle in a spatially and temporally dynamic fashion (Golsteyn et al., 1995) . It has been suggested that this kinase plays a role in the dynamic function of the mitotic spindle during chromosome segregation (Golsteyn et al., 1995). Since the MPM-2 antibody stains components of the mitotic spindle 18 (Vandre et al., 1984) in a pattern similar to the localization of Plk 1, there is a possibility that spindle-associated Plk 1 may phosphorylate MPM-2 reactive proteins involved in the regulation of spindle function.
The first part of this dissertation is focused on the investigation of HeLa cell MAP4 kinase activity that phosphorylates MAP4 in a cell cycle dependent fashion, especially at the MPM-2 epitope site(s). Both the endogenous kinase activities present in mitotic HeLa samples and candidate exogenous sources of MPM-2 epitope kinases, such as p34cdc2, MAP kinase, casein kinase-2, and
Plk 1 were examined. In the second part, the potential MPM-2 epitope on MAP4 was investigated by peptide mapping of mitotic MAP4 using chemical cleavage or by limited proteolytic digestions of mitotic MAP4. CHAPTER II
MATERIALS AND METHODS
M a t e r i a l s
Cell culture medium was obtained from BioWhittaker and was supplemented with calf serum (Intergen Company). Nocodazole, hydroxyurea, aphidicolin, myelin basic protein, casein, N-chlorosuccinimide (NCS), Protein A- agarose, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease were all purchased from Sigma Chemical Company, while histone Hi and endoproteinase Arg-C (sequencing grade) were obtained from Boehringer Mannheim Biochemicals. Goat anti-rabbit immunobeads and peroxidase-conjugated secondary antibodies were from Hyclone. The Western blots were developed using the
LumiGLO™ chemiluminescent substrate kit (Kirkegaard &
Perry Laboratories, Inc.) or 4-chloro-l-napthol (Sigma chemical company). Purified p44mpk (MAP kinase), cdc2
(p34cdc2) , and casein kinase-2 (CK-2), anti-rat MAP
19 20 kinase, and anti-human cdc2 were obtained from Upstate Biotechnology, Inc., while anti-human cyclin A and cyclin
Bl antibodies were obtained from Oncogene Science. 32p- orthophosphate and [y-32P] ATP were purchased from ICN
Radiochemical. Protein determinations were carried out with the BCA assay (Pierce Chemical Company).
Tissue culture and metabolic labeling
Hel.a human epitheloid carcinoma cells of the S3 strain were grown in suspension culture in 3 liter spinner flasks at 37 eC. The cells were cultured in Joklik-modified Minimum Essential Medium supplemented with 10% iron- enriched calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin and 2 mM glutamine, and cells were grown to a final density of 6 x 105 cells/ml. Normally, 7 liters of liquid culture were harvested for a single preparation of HeLa microtubule protein.
Monolayer cultures of HeLa cells were maintained in Ham's F-10 medium supplemented with 10% fetal bovine serum and antibiotics as above. For synchronization of monolayer cultures, the cells were blocked in thymidine (2-5 mM) for 14-16 hr, released from the block by incubation in Ham's F-10 medium for 9-10 hr, then synchronized at the Gl/S phase boundary with aphidicolin (5 p.g/ml) for an additional 12-14 hr. The majority of cells were observed 21 to be in mitosis 11-13 hr after release from the aphidicolin block as determined by phase contrast microscopy. Prior to metabolic labeling with 32p- orthophosphate, cells were incubated in phosphate-free Ham's supplemented with dialyzed fetal bovine serum containing 100-500 |iCi/ml 32P-orthophosphate for 1 hr.
(The aphidicolin synchronization studies were carried out by Mary Bassett.)
Preparation of microtubule protein from synchronized HeLa cells
For the preparation of mitotic microtubules nocodazole (0.04 |ig/ml) was added to the culture medium, and for the preparation of interphase microtubules hydroxyurea (2.5 mM) was added to the medium respectively 16-20 hr prior to harvesting the cells. Taxol-stabilized microtubules were prepared from the synchronized cell populations as described by Vallee and Collins (1986), and the purification procedure is summarized in Table 1. Briefly, cells were collected by spinning at 2500 rpm in a JA-14 rotor (Beckman Instrument Inc.) at 4 eC for 5 min. Pelleted cells were washed in ice cold PBS (140 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HP04, 1.5 mM KH2P04) followed by PEM (0.1 M PIPES-NaOH, pH 6.6 + 1 mM EGTA + 1 mM MgS04) buffer and swollen in swelling solution (1 mM EGTA + ImM MgS04, pH 6.6). Swollen cells were lysed using a Dounce-type tissue homogenizer with the tight-fitting pestle. High speed soluble cytoplasmic supernatant was collected by centrifugation at 45,000 rpm with a Ti-60 rotor (Beckman Instrument Inc.) at 4 9C for 90 min. Taxol and GTP were added (20 |JM and 1 mM, respectively), and the supernatant was warmed to 37 eC for 10 min to induce polymerization of tubulin. Polymerized microtubules were pelleted by centrifugation at 18,000 rpm with a JA-20 rotor (Beckman) at 25 9C for 30 min. Fractions obtained from the HeLa cell preparations including whole cell lysate, soluble cytoplasmic supernatant, microtubule-depleted supernatant, and microtubule pellets were used for kinase assays (see below).
Purification of MAP4 and 125 kD MAP from microtubule protein
Microtubule pellets prepared as above were resuspended in 500 (ll of PEM buffer containing 0.35 M NaCl, boiled for 5 min, and centrifuged at 10,000xg for 10 min at 4 eC. The supernatant was recovered as a heat-stable MAPs fraction, which primarily contained both MAP4 and 125 kD MAP.
Preparation of whole cell lysates and immunoprecipitation of MAP4
Prior to extraction of monolayer HeLa cultures, attached cells were rinsed with cold phosphate-buffered saline 23 (PBS) (140 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HP04, 1.5 mM KH2P04). After addition of RIPA lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.05% SDS, 0.1 M Na2HP04 (pH 7.2), 10 mM EDTA, 10 mM EGTA, 10 mM NaF, 0.1 mM Na3V04, 100 KIU aprotinin/ml, 1 mM PMSF, 1 |ig/ml leupeptin, 1 |ig/ml pepstatin) to the culture dish, the cells were removed with a rubber policeman and placed on ice for 10 min. Whole cell lysates (200 Jig protein in 500 |ll RIPA buffer) were precleared with 25 Jll of non-immune rabbit serum for 1 hr on ice. MAP4 was immunoprecipitated using 5-10 |i.l of rabbit polyclonal HeLa MAP4 serum. Immune complexes were collected following incubation with agarose-linked goat anti-rabbit igG, the immunobeads were then washed in 4 rinses of RIPA, and heated in SDS sample buffer without mercaptoethanol. The supernatant was analyzed by SDS-PAGE, and autoradiographic bands were quantitated using an Optimas based image analysis system.
Ixnmunoblott ing
The boiled microtubule samples were subjected to 7% SDS- PAGE, transferred to nitrocellulose and analyzed by Western blots. The nonspecific binding sites on the nitrocellulose membrane were blocked with 10% heat- inactivated horse serum in Tris-buffered saline (TBS) (0.9% NaCl, 10 mM Tris-HCl, pH 7.4). Blocked membrane was incubated with primary antibody for 2 hr at room temperature, rinsed with TBS, and incubated with 24 peroxidase-conjugated secondary antibody for 1 hr at room temperature. After washing in TBS for 30 min at room temperature, the bound secondary antibody was visualized with 4-chloro-l-naphthol or by chemiluminescent detection system. MAP4 was detected using two different antibodies; rabbit polyclonal anti-MAP4 (Bulinski and Borisy, 1980a) and phosphoprotein specific mouse monoclonal MPM-2 (Davis et a l ., 1983). 125 kD MAP was detected with a rat polyclonal antibody (Vandre, unpublished results). Western blot analysis of mitotic HeLa microtubule was performed as above using the following primary antibodies; anti-MAP4, MPM-2, anti-125 kD MAP, anti-cyclin A, anti- cyclin B, anti-rat MAP kinase, and anti-human cdc2. 10% acrylamide gels were used for cdc2, MAP kinase, cyclin A, and cyclin B immunoblot analysis. Interphase HeLa MAP4 was examined by MPM-2 immunoblot following phosphorylation with purified kinase preparations or endogenous kinase activities present in mitotic HeLa samples as above.
Protein kinase assays
Protein kinase assays were performed essentially as described by Tombes et al (1991). For endogenous kinase assay of HeLa mitotic microtubule preparations, 10 ^g of microtubule suspension was incubated in a final volume of 30 |il buffer A containing 30 mM HEPES (pH 7.4), 10 mM
MgCl2, 1 mM DTT, 20 mM P-glycerophosphate, 1 mM EGTA, 0.1 mM Na3V04, 1 mM NaF, and 0.5 mM cAMP-dependent protein 25
kinase inhibitor peptide in the presence of 5 |lCi [y-32P]
ATP. The reaction was performed at 30 9C for 15 min and stopped by adding 5x concentrated Laemmli's SDS sample buffer (1970) and analyzed by 7% SDS-PAGE. Subsequently, the proteins were transferred to nitrocellulose, followed by autoradiography. Kinase assays for mitotic HeLa samples (whole cell lysate, soluble cytoplasmic supernatant, microtubule-depleted supernatant, and microtubule pellets) were performed using P81 phosphocellulose paper. The assays were carried out as above except the reactions were terminated by spotting and drying the reaction mixtures on P81 phosphocellulose paper. The papers were washed with 75 mM phosphoric acid 5 times for 10 min each and the phosphate incorporated into protein was counted using a Beckman LS 7800 scintillation system. The exogenous purified kinases p44mpk, p34cdc2 kinase, and casein kinase-2 (25 ng each) were used to phosphorylate heat-stable HeLa MAPs (2 |ig) isolated from either synchronized mitotic or interphase cells as above.
Fractionation of kinase activities from mitotic
HeLa microtubule-depleted supernatant by ammonium sulfate precipitation
The proteins in the mitotic HeLa microtubule-depleted supernatant were precipitated into 0-25%, 25-35%, 35-45%, and 45-100% (NH4)2S04 fractions. Solid (NH4)2S04 was added to the mitotic HeLa microtubule-depleted supernatant with constant stirring at 4 9C. When all of the salt was added and completely dissolved, the mixture was centrifuged at 10,000xg for 15 min. The supernatant fluid was decanted and used as the starting material for the next fractionation. The pellet was dissolved in PEM buffer and dialyzed against PEM buffer at 4 9C. The kinase activities present in each (NH4)2S04 fraction were assayed using histone Hi or MAP4 as a substrate. The assays were carried out as above using P81 phosphocellulose paper and autoradiography was performed following 7% SDS-PAGE.
Immunoprecipitation and protein kinase assay of
Plk 1
The rabbit immune serum (R32) which was raised against the COOH-terminal 201 amino acids of human Plk 1 was generously provided by Dr. E.A.Nigg. The immunoprecipitation of Plk 1 and kinase assay of immunoprecipitates were performed as described (Golsteyn et al., 1995).
R32 serum (at 1:100; vol/vol) was added to soluble cytoplasmic supernatant of mitotic HeLa cell extract and the sample was incubated on ice for 1 hr. Protein A- agarose beads was added to sample and immune complex was 27 collected after 30 min incubation at 4 2c. Bead buffer (50 mM Tris, pH 7.5, 0.1% NP-40, 250 mM NaCl, 5 mM NaF, 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl phosphate, 1 |ig/ml leupetin, and 1 ng/ml pepstatin) was used to wash the beads three times.
To analyze the immune complexes by SDS-PAGE, washed beads were transferred to new tubes, SDS sample buffer was added and boiled for 5 min at 95 9C. The localization of human Plk 1 in mitotic HeLa cells was examined by Western blot analysis of mitotic HeLa cell fractions (whole cell lysate, soluble cytoplasmic supernatant, microtubule- depleted supernatant, and microtubule pellet). The presence of MAP4 in the Plk 1 immunoprecipitate was checked by immunoblot analysis of Plk 1 immunoprecipitate probed with polyclonal anti-MAP4 antibody.
For kinase assay of immunoprecipitates, beads were washed in Plk 1 wash buffer (20 mM HEPES, pH 7.4, 150 mM KCl, 10 mM MgCl2» 1 mM EGTA, 0.5 mM DTT, and 5 mM NaF) and stored on ice until used. To start the kinase assay, 20 |il of Plk 1 assay buffer (Plk 1 wash buffer + 10 |iM ATP + 4 |XCi of [y-32P] ATP (10 mCi/ml), and 0.5 mg/ml of dephosphorylated casein or 2 |j.g of mitotic or interphase HeLa MAP4) was added to the immunoprecipitate and incubated for 30 min at 30 9C. Reactions were stopped by adding equal volume of 2.5x SDS sample buffer and boiled for 5 min at 95 9C. The samples were analyzed by SDS-PAGE 28 followed by autoradiography.
Peptide mapping
The peptide mapping of mitotic MAP4 with N-chlorosuccinimide (NCS) was performed as described (Goding, 1986). Mitotic MAP4 sample was run on 7% SDS- PAGE and stained with coomassie blue. The MAP4 band was cut out from the gel and rehydrated in acetic acid/urea/water (AUW, 10 ml glacial acetic acid/10 g urea/10 ml H 20) for 10 min at room temperature with occasional agitation. The gel slice was transferred to NCS solution (20 mg NCS per 10 ml AUW) and rotated for 20 min at room temperature. The NCS solution was removed, and 10 ml of 1.0 M Tris-HCl, pH 8.0 was added to the gel slice to neutralize acid. The sample was rotated an additional 10-20 min. The liquid was aspirated, replaced with SDS sample buffer, and heated to 95 9C. Subsequently, the gel slice was pushed into the well of r the stacking gel of a new gel and overlaid with sample buffer. The new 7% SDS-PAGE was run, proteins were transferred to nitrocellulose, and an MPM-2 immunoblot analysis was performed.
Peptide mappings of mitotic HeLa MAP4 by limited proteolysis were performed using trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Arg-C. Proteolytic enzyme was added to the mitotic HeLa 29 MAP4 (enzyme:substrate = 1:100; w/w) and incubated for various periods of time at 37 9C. Reactions were terminated by adding 100 mM PMSF to a final concentration of 2 mM, and SDS sample buffer was added, followed by heating to 95 eC for 5 min. MAP4 digests were subjected to 4-12.5% gradient SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blot analysis. Rabbit polyclonal anti-MAP4 (Bulinski and Borisy, 1980a) and MPM-2 (Davis et al., 1983) were used to probe MAP4 proteolytic digests.
For the sequencing of MAP4 peptide fragments, MAP4 was incubated with endoproteinase Arg-C (enzyme:substrate = 1:50; w/w) for 2 hr or with V8 protease for 20 min and run on 4-12.5% gradient SDS-PAGE. Subsequently, the digested samples were transferred to polyvinylidene difluoride (PVDF) membrane. The procedure for the sequence analysis of protein electroblotted onto PVDF membrane is described by Matsudaris (1987). After transfer, the PVDF membrane was washed in deionized water for 5 min, stained with 0.1% Coomassie Blue R-250 in 50% methanol for 5 min, and then destained in 50% methanol, 10% acetic acid for 10 min at room temperature. Finally, the PVDF membrane was rinsed in deionized water for 10 min, air dried, and stored at -20 eC. The coomassie blue stained MAP4 peptide fragment which has MPM-2 reactivity generated by endoproteinase Arg-C and MPM-2 non-reactive MAP4 peptide fragment produced by V8 protease were cut out with a clean razor 30 and the N-terminal 10 amino acid residues were determined by John Lowbridge at the Biochemical Instrument Center.
Microtubule binding assay of mitotic HeLa MAP4 peptide fragments generated by V8 protease digestion
Mitotic HeLa MAP4 was digested by V8 protease as above and incubated with purified rat brain tubulin (generously provided by Min Ding) in the presence of taxol (20 |iM) and GTP (1 mM) at 37 9C for 20 min. The sample was centrifuged at 10,000xg for 20 min, and the supernatant and pellet, which was resuspended in a volume of PEM buffer equal to that of the supernatant, were subjected to 7% SDS-PAGE followed by immunoblot analysis using MPM-2 antibody. The same assay was performed with undigested MAP4 as a control. CHAPTER III
RESULTS
Analysis of heat stable MAPs from interphase and mitotic HeLa cells.
To purify MAP4 and 125 kD MAP from interphase and mitotic HeLa cells, NaCl was added to 0.35 M to taxol-stabilized microtubules followed by boiling for 5 min. The thermostable MAP fraction was recovered in the supernatant after denatured tubulin was removed by centrifugation at 10,000xg for 10 min at 4 9C (Figure 1A) . From a 7 liter culture of HeLa cells approximately 100 ng of heat-stable MAPs were obtained, with a purity of >90%. In addition to the major MAP4 coomassie stained band, two other heat stable MAP bands were stained, which corresponded to a 255 kD MAP4-related protein (Bulinski and Borisy, 1980b) and the 125 kD MAP called ensconsin (Bulinski and Bossier, 1994). It should be noted that both the 255 kD band and MAP4 present in the mitotic sample (MS) were shifted to a higher apparent molecular weight as compared to the
31 interphase heat stable MAP sample (IS). A significant amount of 125 kD MAP was also present in the mitotic HeLa MAP sample. Immunoblot analysis of MAP4 and 125 kD MAP obtained from interphase and mitotic HeLa cells are shown in Figure IB, C, and D. The MAP4 polyclonal antibody recognizes both interphase and mitotic HeLa MAP4, and once again the molecular weight shift of mitotic MAP4 was observed (Figure IB). The MPM-2 phosphoepitope specific antibody recognized only mitotic HeLa MAP4 (Figure 1C) indicating that the molecular weight shift of MAP4 observed in mitotic samples correlated with mitosis- specific phosphorylation of MAP4. 125 kD MAP antibody staining showed that this MAP was enriched in the heat stable MAP fraction prepared from mitotic microtubules (Figure ID).
Cell cycle dependent phosphorylation of MAP 4
To further demonstrate the correlation between the MPM-2 recognition and the phosphorylation state of mitotic MAP4, we examined the incorporation rate of 32P-orthophosphate into MAP4 in vivo. HeLa cells were synchronized at the Gl/S boundary with aphidicolin, and at various times following release from the block, the cells were labeled with 32P-orthophosphate for 1 hr. Immunoprecipitates of
MAP4 were prepared from whole cell lysates and analyzed following separation by polyacrylamide gel electrophoresis (Figure 2). While it is clear that the amount of MAP4 did not change over the course of the experiment (Figure 2A) , an increased rate of phosphate incorporation was observed beginning 10 hr following release of the aphidicolin block (Figure 2B). A high rate of phosphate incorporation continued over the next few hours, but decreased 14-16 hr following release. The increased rate of phosphate incorporation from 11-13 hr corresponded to the period of time in which most of the cells were in mitosis. The subsequent reduction in incorporation occurred as the population returned to interphase. The rate of phosphate incorporation increased nearly 7.5 fold as determined by densitometric quantitation of the autoradiogram (Figure 2C). In nocodazole blocked mitotic HeLa cells, the phosphate label incorporated into MAP4 continually turns over with a half-life of approximately 4 hr; suggesting that both MAP4 kinase and MAP4 phosphatase activities are present in mitotic cells (data not presented). In addition, the phosphate label incorporated into mitotic MAP4 is rapidly lost following release of the nocodazole block, and entry of the cells into G1 and interphase (data not presented). These initial metabolic labeling studies were carried out by Mary Bassett, and confirmed that a cell-cycle dependent phosphorylation of MAP4 occurred in 34
la vitro phosphorylation of MAP4 by exogenous
kinases.
The MPM-2 antibody recognizes mitotic phosphoproteins in every mammalian species examined (Vandre et a l ., 1986), and mitotic MAP4 in both CHO and HeLa cells (Vandre et al, 1991). Therefore, the MPM-2 epitope on MAP4 is expected to be conserved among mammalian species. As an initial step towards identifying both the phosphorylation sites on MAP4 that are recognized by the MPM-2 antibody and determining the kinase(s) that phosphorylates that site or sites, we examined the nucleotide sequences that have been determined for human, mouse, and bovine MAP4 (West et al., 1991; Aizawa et al., 1990) for the presence of consensus
phosphorylation sites for p34cdc2, MAP kinase, and casein
kinase-2. While each MAP4 sequence has a number of potential proline-directed serine/threonine phosphorylation sites, only two of these are conserved in all three MAP4 sequences. Similarly, several potential casein kinase-2 sites are also present, but only three are conserved. The conserved consensus phosphorylation sites located on MAP4 are presented in Table 2. Therefore, we examined the ability of these three selected kinases to phosphorylate isolated HeLa MAP4 in vitro.
Isolated mitotic HeLa MAP4 was incubated with exogenous kinases; p44mpk, p34cdc2, and casein kinase-2 (CK-2), and 35 autoradiographs of the reaction products were prepared after the samples had been separated by SDS-PAGE (Figure 3A). Control samples without added MAP4 showed that
autophosphorylation of p44mpk and p34cdc? occurred (Figure
3A, lanes 1 and 3). When the heat stable HeLa mitotic
MAP4 sample was added, only p44mpk and p34cdc2 were
capable of phosphorylating MAP4 and the 255 kD MAP (Figure 3A, lanes 4 and 6). The mitotic MAP4 did not serve as a substrate for casein kinase-2, however, the 125 kD MAP was readily phosphorylated (Figure 3A, lane 5). To examine the possibility that phosphorylation sites preexisting from in vivo mitotic phosphorylation of MAP4 prevented labeling, we examined whether interphase MAP4 served as a substrate for the purified kinases (Figure 3B). Once again, interphase MAP4 was phosphorylated by p44mpk and
p34cdc2, but was not phosphorylated by casein kinase-2
(Figure 3B, lane 2). Since the casein kinase-2 was active, as indicated by its ability to phosphorylate 125 kD MAP, it remains possible that interphase MAP4 contains endogenous phosphate residues that precluded casein kinase-2 phosphorylation of the protein. We have not attempted to dephosphorylate the purified MAP4 prior to in vitro kinase incubation to examine this possibility. It was clear, however, that the potential endogenous phosphorylation sites present on interphase MAP4 were not associated with the mitosis specific MPM-2 phosphoepitope, since interphase MAP4 is not recognized by the MPM-2 36 antibody (Figure 1).
Endogenous HeLa cell MAP4 kinases are enriched in microtubule-containing samples.
We examined whether endogenous MAP4 kinase activities related to p34cdc2 or MAP kinase were present in synchronized populations of mitotic HeLa cells, and/or associated with microtubule-containing structures from HeLa cells. Kinase activities were assayed in various HeLa cell fractions towards exogenous substrates such as histone Hi, myelin basic protein, or casein in addition to endogenous substrates present in the mitotic HeLa sample (Table 3). Mitotic HeLa cells were fractionated into total lysates, soluble cytoplasmic supernatant, microtubule-depleted supernatant, and microtubule pellet. It should be noted that all of the kinase activities assayed were enriched in the microtubule pellet when compared to the other samples.
Endogenous protein substrates phosphorylated by kinases associated with mitotic microtubules were visualized by autoradiography after 7% SDS-PAGE (Figure 4A). 255 kD MAP, MAP4, 125 kD MAP, and several other proteins were phosphorylated by endogenous mitotic HeLa cell microtubule associated kinases. Immunoblots of the mitotic microtubule pellets are shown in Figure 4B. 37 MAP4, 125 kD MAP, and MPM-2 reactive proteins were clearly
detected as expected. MAP kinase, p34cdc2, and cyclin B
were also shown to be associated with the mitotic HeLa microtubule preparations as indicated by the immunoblot analysis, but little or no cyclin A was detected (Figure 4B) .
Interphase HeLa MAP4 served as a substrate for the endogenous mitotic kinase activities (Figure 5). The autoradiogram showed that both mitotic microtubules and microtubule-depleted supernatant had MAP4 kinase activity. Phosphorylation of endogenous mitotic MAP4 was also observed in the microtubule pellet sample (Figure 5, lane 1) . In addition, it should be noted that only 1 fig'of microtubule pellet was used in comparison to 10 ^.g of microtubule-depleted supernatant to detect the kinase activity presented in Figure 5. Thus, the MAP4 kinase(s) were clearly enriched in the microtubule pellet when compared with the microtubule-depleted supernatant.
In order to optimize the reaction conditions for the MAP4 kinase activity and gain some initial insight into the properties of the MAP4 kinase, mitotic HeLa microtubules samples were assayed using several different buffer conditions (Figure 6); including buffers designed for casein kinase-2 (Litchfield et al., 1990), protein kinase C (Go et al., 1987), cAMP-dependent protein kinase 38
(Roskoski, 1983), or p34cdc2 kinase (Tombes et a l ., 1991).
The results obtained indicated that the MAP4 kinase activity in the microtubule pellet was maximal in buffer
conditions that were favorable for p34cdc2 kinase (Figure
6). Similar results were obtained with the microtubule- depleted supernatant. Phosphorylation of interphase MAP4 by mitotic microtubule-associated kinases was performed with varying concentrations of interphase MAP4 to determine the optimal substrate concentration for the kinase assays. Maximal incorporation of phosphate into MAP4 occurred when 0.5 (ig of MAP4 was added to 1 |J.g mitotic microtubule pellet (Figure 7, lane 3). An inhibition of kinase activities was observed with increasing amount of MAP4, suggesting that excess substrate may titrate out kinase activity, perhaps by binding of kinase to the MAP4 forming a complex that was less active.
Phosphorylation of interphase MAP 4 converts the protein into an MPM-2 reactive form.
Using an in vitro assay system, p34cdc2 was shown to be an
efficient exogenous MAP4 kinase. Therefore, we determined whether the phosphorylation of interphase MAP4 by exogenous p34cdc2 could also generate a phosphoepitope recognized by the MPM-2 monoclonal antibody. The phosphorylation of interphase HeLa MAP4 by p34cdc2 kinase 39 showed increased 32P incorporation with respect to time
(Figure 8A) . Following p34cdc2 phosphorylation, the interphase MAP4 was converted to an MPM-2 reactive form, however, this occurred only after extended incubation times (Figure 8B). in several different experiments, MPM- 2 reactivity was never detected prior to 2-4 hr of reaction with the p34cdc2 (data not shown) .
Interphase MAP4 was also phosphorylated by the kinase activities present in mitotic HeLa microtubule pellet samples (Figure 9A), and this phosphorylation also generated an MPM-2 reactive form of interphase MAP4 (Figure 9B). The endogenous MAP4 present in the mitotic microtubule sample may have partially contributed to the phosphate that was incorporated during the kinase reaction. In addition, the endogenous mitotic MAP4 was already MPM-2 reactive prior to the in vitro phosphorylation reaction. The preexisting mitotic MAP4 made interpretation of the results from this experiment more complex. However, it was clear that increased incorporation of phosphate was obtained in the MAP4 region of the gel in samples where interphase MAP4 was added, and additional MPM-2 reactive bands were also obtained in these samples when probed with MPM-2 antibody. Importantly, the generation of the MPM-2 epitope on interphase MAP4 by endogenous microtubule-associated kinases differed from that obtained after phosphorylation 40
with exogenous p34cdc2 in that the endogenous microtubule-
associated kinase activity was capable of forming the MPM- 2 epitope much more rapidly. It should also be noted that conversion of the interphase MAP4 to an MPM-2 reactive form did not cause the molecular weight shift common to the endogenous mitotic forms of MAP4.
We attempted to fractionate the MAP4 kinase activity that was associated with the mitotic microtubule pellet. Following salt extraction of the microtubule pellets, MAP4 kinase activity was shown to be present in the supernatant (Figure 10A). Unexpectedly, kinase activity was also shown to remain in the salt extracted microtubule pellet even though all of the endogenous MAP4 was removed from the microtubules following salt extraction. Neither the kinase activity present in the salt extract, nor the kinase activity in the extracted microtubule pellet, was capable of converting the interphase MAP4 into an MPM-2 reactive form, however (Figure 10B). We have been unable to reconstruct the MPM-2 kinase activity by recombining the salt extract with the extracted microtubules (data not presented). It is possible that phosphatases also associated with the microtubules are also removed by the salt extraction, and these may inactivate the MPM-2 kinase. We have not exhaustively explored this possibility. Although MAP4 is thermostable, boiled MAP4 might be partially denatured, and thus, structurally altered from the native protein. If boiling induced some conformational changes, this could result in conversion of MAP4 into a form that was not an appropriate kinase substrate. To investigate this possibility, we compared three different preparations of interphase MAP4; MAP4 prepared in association with the interphase microtubule pellet, MAP4 dissociated from interphase microtubules by salt extraction, and MAP4 obtained by boiling the interphase microtubules as described above. Each of these samples of interphase MAP4 were examined as substrates for in vitro phosphorylation by endogenous kinase activity present in the mitotic HeLa microtubule-depleted supernatant. Although the overall MAP4 kinase activity was lower in the microtubule-depleted supernatant, these samples were essentially free of endogenous mitotic MAP4 that was MPM-2 reactive.. Varying amounts of other unidentified MPM-2 reactive mitotic proteins were present in the microtubule-depleted mitotic supernatants, however. All three forms of interphase MAP4 were substrates for mitotic HeLa kinase(s), and each MAP4 form was converted to an MPM-2 reactive phosphoprotein (Figure 11). Therefore, boiling of the MAP4 apparently had no effect on its ability to be phosphorylated by endogenous kinases, or its ability to gain MPM-2 reactivity. 42 The effect of phosphatase inhibitors on the overall phosphorylation of interphase HeLa MAP4, and its conversion to an MPM-2 reactive form by endogenous mitotic kinases was also examined (Figure 12). Again, the microtubule-depleted supernatants were utilized to eliminate endogenous mitotic MAP4. The overall kinase activity of the mitotic microtubule-depleted supernatant was higher in the presence of microcystin, but the generation of the MPM-2 reactive epitope was similar to samples reacted in the absence of microcystin. Similarly, increased MPM-2 reactivity was not observed in MAP4 samples reacted in the presence of okadaic acid. This suggests that the MPM-2 phosphorylation site was not overly sensitive to endogenous phosphatase activity present in the mitotic microtubule-depleted supernatant.
Fractionation of kinase activities from mitotic
HeLa microtubule-depleted supernatant by ammonium
sulfate precipitation
Kinase activities present in the mitotic microtubule- depleted supernatant were fractionated by (NH4)2S04 precipitation. A kinase assay of the fractions using P81 phosphocellulose showed that the 25-35% (NH4)2S04 fraction was most enriched in kinase activities, including Hi kinase activities (data not shown). However, the autoradiogram of similar kinase reactions indicates that the 25-35% (NH4)2S04 fraction does not have MAP4 kinase 43 activity. The MAP4 kinase was most enriched in the 35-45% (NH4)2S04 fraction (Figure 13).
Analysis of the distribution of human Plk 1 in mitotic HeLa cells.
Polo, a serine/threonine-specific kinase first identified in Drosophila, is one of the protein kinases implicated in controlling spindle function and chromosome segregation (Llamazares et al., 1991). The S. cerevisiae gene CDC5 was found to encode a polo-related protein kinase (Kitada et al., 1993) and recently, a human protein kinase that displays a substantial degree of sequence identity with Drosophila polo and budding yeast CDC5 have been identified. This human homolog was termed polo-like kinase 1 (Plk 1) (Golsteyn et al., 1994). The activity of Plk 1 isolated from synchronized HeLa cells was found to be low during interphase, but high during mitosis (Golsteyn et al., 1995).
We examined the distribution of human Plk 1 in mitotic HeLa cells by immunoblot analysis of the mitotic HeLa cell fractions. The fractions obtained from the mitotic HeLa cell microtubule preparation including whole cell lysate, soluble cytoplasmic supernatant, microtubule-depleted supernatant, and microtubule pellets were run on 7.5% SDS- PAGE, transferred to nitrocellulose, and probed with R32 rabbit polyclonal anti-Plk 1 (Golsteyn, et al., 1995). 44 The immunoblot shows that Plk 1 was present in all of the fractions, however, Plk 1 was most enriched in the microtubule pellet (Figure 14, lane 4).
Immunoprecipitation and protein kinase assay of
Plk 1
Since Plk 1 is a kinase specifically activated during mitosis (Golsteyn et al., 1995), and it is most enriched in the mitotic microtubule pellet obtained from HeLa cells (Figure 14), there is a possibility that MAP4 may be phosphorylated by Plk 1 during mitosis. We examined this possibility with a in vitro kinase assay using immunoprecipitated Plk 1 and MAP4 as a substrate.
Plk 1 was immunoprecipitated from mitotic HeLa cell soluble cytoplasmic supernatant using the R32 serum (Golsteyn et al., 1995). Western blot analysis of the immunoprecipitate is shown in Figure 15. We subsequently used this immunoprecipitate to perform an in vitro kinase assay. Casein, interphase HeLa MAP4, and mitotic HeLa MAP4 were used as substrates for Plk 1. Since human Plk 1, Drosophila polo, and yeast CDC5 have been reported to phosphorylate casein (Golsteyn et al., 1995; Fenton et al., 1993; Kitada et al., 1993), casein was used as a control for this kinase assay. The autoradiogram of the kinase assay using the Plk 1 immunoprecipitate is shown in Figure 16. No exogenous substrate was added in lane 1, 45 therefore, the labeled proteins are the endogenous substrates which were precipitated with Plk 1. Plk 1 was shown to be active as a casein kinase (lane 2). Both interphase and mitotic HeLa MAP4 were phosphorylated by the Plk 1 immunoprecipitate (lanes 3, 4). Interphase MAP4 was more heavily labeled compared to mitotic MAP4, suggesting that some Plk 1 phosphorylation sites on mitotic MAP4 might have been phosphorylated in vivo preventing further labeling in vitro.
The time course for phosphorylation of interphase HeLa MAP4 by Plk 1 shows that the labeling increases with respect to time, and was saturated after about an 1 hr incubation (Figure 17). We also examined whether Plk 1 could convert interphase MAP4 into an MPM-2 reactive form, however, the immunoblot showed the Plk 1 is not the MPM-2 epitope kinase of MAP4 (data not shown).
Since MAP4 is a good substrate for Plk 1, and Plk 1 is associated with microtubules, we examined the possible association of MAP4 with Plk 1. Plk 1 was immunoprecipitated from mitotic HeLa cytosolic supernatant and immunoblot analysis was performed with polyclonal MAP4 antibody. The result shows that MAP4 did not coprecipitate with Plk 1 (Figure 18). 46
Peptide mapping of mitotic HeLa MAP4.
As mentioned above, one of our long-term goals is to determine the MPM-2 epitope site on MAP4. In order to achieve this goal, we performed peptide mapping of mitotic HeLa MAP4 using chemical cleavage or proteolytic enzymes. The basic strategy for this study was to generate an MPM-2 reactive MAP4 peptide for sequence analysis.
N-chlorosuccinimide (NCS) was used to cleave MAP4 at the tryptophan residues. Human MAP4 has only 3 tryptophan residues, and these residues are located at amino acid positions 19, 190, and 322. The complete sequence of MAP4 contains 1152 amino acid residues. Therefore, the cleavage of MAP4 with NCS is expected to generate three small peptides and one large fragment. The size of the peptide fragments would be as follows : the 19 amino acid residues at the N-terminus, a 171 amino acid residue fragment (residues 20-190), a 131 amino acid residue fragment (residue 191-322), and the largest fragment containing 829 amino acids (residue 323-1152). Our result shows that cleavage of mitotic MAP4 with NCS generated a single fragment of 150-175 kD which is MPM-2 reactive (Figure 19), indicating that the MPM-2 epitope of MAP4 is not located at the first N-terminal 322 amino acids.
The peptide map of MAP4 generated by trypsin digestion is shown in Figure 20. The cleavage sites of trypsin are on 47 the carboxy side of arginine or lysine residues, and MAP4 has a number of potential cleavage sites. Therefore, a limited trypsin digestion was performed. Rabbit polyclonal anti-MAP4 and MPM-2 antibody were used to probe the peptides that were generated. The MPM-2 epitope was very quickly destroyed by trypsin. After only a 2 min digestion, a peptide that was weakly MPM-2 reactive around 50 kD was detected, but this peptide was destroyed after further digestion. Chymotrypsin was also used to generate a MAP4 peptide map (Figure 21). Chymotrypsin cuts the protein on the carboxy side of tyrosine, tryptophan, phenylalanine, and leucine residues. Limited digestion of mitotic MAP4 by chymotrypsin shows a fragment about 40 kD that is MPM-2 immunoreactive after a 20 min digestion. As a third proteolytic enzyme, V8 protease was used. This enzyme cuts on the carboxy side of glutamic acid or aspartic acid. V8 protease also generated MPM-2 reactive MAP4 fragments around 40 kD after a 20 min digestion (Figure 22).
We attempted to determine the amino acid sequence of the smallest MPM-2 reactive V8 protease fragment, however, we were not able to detect a coomassie stained band corresponding to this fragment on the PVDF membrane. Sequence analysis required at least enough protein to generate a coomassie stained band. However, we were able to determine the N-terminal amino acid sequence of a 30 kD fragment of MAP4 generated by V8 protease which was not 48 MPM-2 reactive. The sequence of this peptide was Lys-Met- Ala-Tyr-Gln-Glu-Tyr-Pro-Asn. The first amino acid determined from this peptide is located at amino acid residue 108 of MAP4. Thus, this result combined with the NCS cleavage result, indicated that we could eliminate an N-terminal region of MAP4 containing the first 400 amino acid residues as the region containing the potential MPM- 2 epitope.
The microtubule binding properties of MAP4 peptide fragments generated by V8 protease were examined (Figure 23). After digestion of MAP4 with V8 protease, purified taxol-stabilized rat brain tubulin was added to the sample and incubated under microtubule polymerization conditions. The microtubule-binding and nonbinding fragments of MAP4 peptides were separated by centrifugation and analyzed by immunoblot analysis. During the incubation with tubulin it was noted that the V8 protease activity was not completely inhibited following addition of PMSF. This resulted in further digestion of MAP4 when the sample was incubated with tubulin. This resulted in similar V8 digestion patterns of MAP4 being observed following incubation with the tubulin fraction regardless of the initial period of MAP4 digestion with V8 protease (data not presented). Stronger protease inhibitors such as APMSF or AEBSF were also used to inhibit V8 protease activity, however, further digestion was still observed. Nevertheless, two MPM-2 reactive MAP4 peptide fragments 49 around 40 kD were observed to be very stable to further proteolysis, and these fragments did not bind to microtubules. This result suggests that the MPM-2 epitope resides on a region of MAP4 that is not an integral part of the microtubule binding domain.
Endoproteinase Arg-C was also used to generate a peptide map of mitotic MAP4 (Figure 24). This enzyme is a cysteine protease which cleaves peptide bonds specifically at the C-terminal side of arginine residues. The smallest MPM-2 reactive MAP4 fragment generated following digestion by endoproteinase Arg-C was about 50 kD in size. This fragment stained with coomassie blue on PVDF membrane, was cut out, and sequence analysis of N-terminal 10 amino acid residues was performed. The sequence determined was Asp- Met-Thr-Leu-Pro-Pro-Glu-Thr-Asn and the first amino acid of this peptide is located at amino acid residue 449 of MAP4. Based upon the size of the MPM-2 reactive peptide this peptide would be approximately 430 amino acids long. Thus, the MPM-2 epitope is localized between amino acid 449 to 880. The MPM-2 epitope of MAP4 is, therefore, located in the proline-rich region of the molecule. This region also contains both conserved consensus sites for cdc2 kinase. These results are consistent with the ability of p34cdc2 kinase to generate the MPM-2 epitope on
MAP4 in vitro. 50
Table 1. Purification of microtubules and MAP4 from HeLa cells.
7 liters of HeLa cells in suspension culture Synchronization (o.o4 |ig/ml nocodazole for mitotic or 2.5 mM hydroxyurea for interphase cells) Pellet, wash, and swell the cells
Dounce homogenize Low speed centrifugation Supernatant , Centrifuge at 18 K rpm Supernatant Centrifuge at 45 K rpm Supernatant Add taxol+GTP and warm to 37 9C '"'entrifuge Microtubule-depleted supernatant Microtubule pellet Add NaCl to 0.35 M and boil for 5 min. Centrifuge Supernatant (Heat-stable MAPs) 51 Table 2. Potential phosphorylation sites are present on and conserved between different MAP4s. Conserved consensus motifs for cdc2(S/TPXK), ERK(PS/TPXK or PXS/TPXK), and casein kinase-2(S/TXXE) are common between human, mouse, and bovine MAP4, and could provide the sites shown to be phosphorylated in HeLa samples by endogenous microtubule associated kinases.
SBg-C.ig£- Residue# Sequence Site #1 human 421 LLSEIEVA casein kinase-2 mouse 381 SLSEIEEA bovine 386 SLSEIEAP site #2 human 438 LSSETEVA casein kinase-2 mouse 406 WSETEW bovine 412 LSSETEVA
Site #3 human 578 PLSETEAT casein kinase-2 mouse 504 PLSEEEVT bovine 527 KFSEAEW
Site #4 human 694 PPSPEKKT p34cdc2 and mouse 665 PPSPEKKA ERK bovine 641 PPSPEKKT
Site #5 human 785 RASPSKPA p34cdc2 mouse 758 PTSPSKPS bovine 732 RVSPSKPA 52 Table 3. Fractionation of microtubule-associated kinases from mitotic HeLa cells.
Fraction Substrate Ha pmole/min/fla Fold ourif Lysate None 12571 317 1 HI 528 1 MBP 405 1 Casein 239 1 Supernatant None 3559 192 0.60 HI 291 0.55 MBP 169 0.40 Casein 186 0.77 Microtubule None 3142 119 0.37 -depleted HI 241 0.46 supernatant MBP 115 0.28 Casein 132 0.55 Microtubule None 92 4242 13 HI 29290 55 MBP 15555 38 Casein 7106 30
1 Fractions were obtained from each step of the preparation of taxol stabilized microtubules. ^ Substrates: None=endogenous activity; Hl=histone Hi; MBP=myelin basic protein. 3 The samples were normalized to protein obtained from 108 mitotic HeLa cells. ^ Kinase activities were measured using P81 phosphocellulose paper. Figure 1. Purification and immunoblot analysis of MAP4 and 125 kD MAP from interphase and mitotic HeLa cells.
Taxol-stabilized microtubules were prepared from synchronized mitotic or interphase HeLa cells as described in materials and methods. Microtubules were resuspended in PEM buffer containing 0.35 M NaCl, boiled for 5 min, and centrifuged at 10,000xg for 10 min at 4 eC. The supernatant containing heat-stable HeLa MAPs and the tubulin pellet were subjected to 7% SDS-PAGE, transferred to nitrocellulose and analyzed by Western blots. MAP4 was detected using two different antibodies: rabbit polyclonal anti-MAP4 and phosphoprotein specific mouse' monoclonal antibody MPM-2. 125 kD MAP was detected with a rat polyclonal antibody.
Samples were as follows: IS, interphase boiled supernatant; IP, interphase boiled pellet; MS, mitotic boiled supernatant; MP, mitotic boiled pellet
Panel A) Coomassie staining of interphase and mitotic HeLa microtubule samples. Panel B) Immunoblot of interphase and mitotic HeLa samples probed with MAP4 antibody. Panel C) Immunoblot of interphase and mitotic HeLa samples probed with MPM-2 antibody. 53 Figure 1 (continued)
Panel D) Immunoblot of interphase and mitotic HeLa samples probed with 125 kD MAP antibody.
The two arrowheads in the MAP4 region indicate mitotic and interphase forms of MAP4: the upper arrowhead for mitotic and the lower arrowhead for interphase MAP4.
54 —?255 kD MAP —MAP4
—125 kD MAP Figure 2. Cell cycle dependent phosphorylation of HeLa MAP4
HeLa cells were synchronized at Gl/S phase and released from the block for times indicated, at which point each
cell sample was labeled with 32P-orthophosphate for 1 hr.
Cells were collected and MAP4 was immunoprecipitated. Panel A) Immunoblot of immunoprecipitated MAP4 indicating little change in total MAP4 protein levels in the cells following release from the Gl/S phase block and progression through mitosis. Panel B) Autoradiogram of immunoprecipitated MAP4 reveals a dramatic increase in incorporation of labeled phosphate beginning 10 hr after release from aphidicolin. The increase in rate of phosphate incorporation was transient, falling to control levels 16 hr after release. The highest rates of incorporation corresponded to passage of the cells through mitosis. Panel C) Quantification of the phosphate incorporated into MAP4 (B), normalized against the MAP4 protein levels (A). Incorporation of labeled phosphate into MAP4 peaked 12 hr after release from aphidicolin, which corresponded to the peak accumulation of mitotic cells as determined by phase contrast microscopy.
56 57
0 3 6 9 10 11 12 13 14 16
B 0 3 6 9 10 11 12 13 14 16 -V i / t v , . ' Sill
0.16
*55 " 0.12
"rau S. 0.08 o ■D0) 0.04 c
0 5 10 15
Release from Aphidicolin (h)
Figure 2 Figure 3. In vitro phosphorylation of MAP4 by exogenous kinases.
Purified p44mpk, p34cdc2, and casein kinase-2(CK-2) were used to phosphorylate interphase and mitotic MAP4. Each kinase (25 ng) was incubated in buffer A (see materials and methods) in the presence or absence of 2 p.g of heat- stable mitotic HeLa MAPs for 15 min at 30 eC (Panel A) . Interphase HeLa MAPs were also used as a substrate for in vitro reaction with the above kinases (Panel B).
Panel A) lanes 1, p44mpk; 2, casein kinase-2;
3, p34cdc2;
4, p44mpk + mitotic HeLa MAPs; 5, casein kinase-2 + mitotic HeLa MAPs;
6, p34cdc2 + mitotic HeLa MAPs
Panel B) lanes 1, p44mpk + interphase HeLa MAPs; 2, casein kinase-2 + interphase HeLa MAPs;
3, p34cdc2 + interphase HeLa MAPs
58 59
Figure 3 Figure 4. Assay of endogenous kinase activity and analysis of proteins associated with mitotic HeLa microtubules.
10 p.g of mitotic HeLa microtubule suspension was incubated in buffer A in the presence of 5 p.Ci [y-32P] ATP for endogenous kinase assay. Proteins associated with mitotic HeLa microtubules were analyzed by immunoblot analysis. 7% SDS-PAGE was used for lanes 1-4, and 10% SDS-PAGE was run for lanes 5-9 (Panel B). Panel A) Autoradiogram of mitotic microtubules following 7% SDS-PAGE. Panel B) Coomassie staining and immunoblots of mitotic microtubules probed with the following antibodies.' lanes 1, Coomassie staining of mitotic microtubule (7% SDS-PAGE); 2, MAP4; 3, MPM-2; 4, 125 kD MAP; 5, Coomassie staining of mitotic microtubule (10% SDS-PAGE); 6, cyclin A; 7, cyclin B;
8, p34cdc2;
9, ERK 60 61
Figure 4 Figure 5. Phosphorylation of interphase HeLa MAP4 by endogenous kinases present in mitotic HeLa samples.
Interphase HeLa MAP4 was phosphorylated by endogenous kinase activities present in mitotic microtubules (1 |J.g) or microtubule-depleted supernatant (10 |lg) in buffer A containing 5 |iCi of [y~32P] ATP. The reactions were performed at 30 SC for 15 min and analyzed by running 7% SDS-PAGE. The proteins were transferred to nitrocellulose, followed by autoradiography. lanes 1, mitotic microtubule; 2, mitotic microtubule + interphase MAP4; 3, mitotic microtubule-depleted supernatant; 4, mitotic microtubule-depleted supernatant + interphase MAP4
62 MAP4
Figure 5 Figure 6. Endogenous kinase activities of mitotic HeLa samples under different buffer conditions.
Endogenous kinase activities present in mitotic HeLa microtubules or microtubule-depleted supernatant were assayed in several different buffer conditions; casein kinase-2 buffer (100 mM Tris-HCl (pH 7.6), 20 mM MgCl2), protein kinase C (PKC) buffer ( 25 mM Tris-HCl (pH 7.5), 6.25 mM MgCl2, 0.125 mM CaCl2, 10 (ig/ml phosphatidylserine, 1 (ig/ml diolein) , cAMP-dependent protein kinase (PKA) buffer (100 mM Tris-HCl (pH 7.5), 20 mM MgCl2, 4 |1M cAMP) , or p34cdc2 kinase buffer (buffer A).
lanes 1-4, endogenous kinase activities of mitotic HeLa microtubule-depleted supernatant in CK-2 (1),
PKC (2), PKA (3), or p34cdc2 kinase buffer
(buffer A) (4); lanes 5-8, endogenous kinase activities of mitotic HeLa microtubules in CK-2 (5), PKC (6), PKA (7), or
p3 4 cdc2 kinase buffer (buffer A) (8).
64 65
MAP4
Figure 6 Figure 7. Effects of MAP4 substrate concentration on the mitotic HeLa microtubule-associated kinase activity
1 |J.g of mitotic HeLa microtubule was incubated with varying amounts of interphase MAP4, and kinase assays were performed. lanes 1, mitotic HeLa microtubules; 2, mitotic HeLa microtubules + 0.25 p.g interphase HeLa MAP4; 3, mitotic HeLa microtubules + 0.5 |ig interphase HeLa MAP4; 4, mitotic HeLa microtubules + 1 (ig interphase HeLa MAP4; 5, mitotic HeLa microtubules + 1.5 (ig interphase HeLa MAP4
66 67
m g l MAP4
Figure 7 Figure 8. Time-course of interphase HeLa MAP4
phosphorylation by p34cdc2 kinase.
Interphase HeLa MAP4 (0.5 |ig) was incubated with p34cdc2 kinase (25 ng) in buffer A containing 5 JlCi [y-32P] ATP for various periods of time (0.5 hr, 1 hr, 2 hr, 4 hr) at 30 9C.
Panel A) Autoradiogram Panel B) MPM-2 immunoblot
68 69
B
0.5 1 2 4 0.5 1 2 4
*-Wvt-\ tv ' *
Figure 8 Figure 9. Phosphorylation of interphase HeLa MAP4 by mitotic HeLa microtubule-associated kinase activities.
Interphase HeLa MAP4 was phosphorylated by the kinase activities present in the microtubule pellet.
Panel A) Autoradiogram lanes 1, mitotic microtubules; 2, mitotic microtubules + interphase MAP4. Panel B) MPM-2 immunoblot same as panel A.
The arrowhead indicates interphase MAP4 which became MPM- 2 immunoreactive following phosphorylation by the kinase(s) associated with mitotic HeLa microtubules (lane
2 ) .
70 71
Figure 9 Figure 10. Phosphorylation of interphase HeLa MAP4 by kinase activities present in mitotic HeLa microtubule salt-extracted supernatant or salt-extracted pellet.
The kinase activities present in mitotic HeLa microtubule pellet were fractionated by salt-extraction. 0.35 M NaCl was added to the mitotic HeLa microtubule suspension, incubated at 37 9C, followed by centrifugation at 10,000xg. The supernatant and pellet fractions were used to phosphorylate interphase MAP4.
Panel A) Autoradiogram lanes 1, mitotic HeLa microtubule salt-extracted supernatant; 2, mitotic HeLa microtubule salt-extracted supernatant + interphase MAP4; 3, mitotic HeLa salt-extracted pellet; 4, mitotic HeLa salt-extracted pellet + interphase MAP4 Panel B) MPM-2 immunoblot lanes are same as panel A.
72 73
Figure 10 Figure 1 1 .Phosphorylation of interphase HeLa MAP4 by mitotic HeLa microtubule-depleted supernatant
The microtubule pellet, microtubule salt-extract, or microtubule boiled supernatant obtained from interphase HeLa cells were used as a substrate for in vitro endogenous kinase activity present in mitotic HeLa microtubule-depleted supernatant.
Panel A) Autoradiogram lanes 1, interphase microtubules control; 2, interphase microtubules + mitotic microtubule- depleted supernatant; 3, mitotic microtubule- depleted supernatant control; 4, interphase microtubule salt-extract control; 5, interphase microtubule salt-extract + mitotic microtubule- depleted supernatant; 6, interphase microtubule boiled supernatant control; 7, interphase microtubule boiled supernatant + mitotic microtubule-depleted supernatant Panel B) MPM-2 immunoblot lanes are same as panel A. The arrowhead indicates interphase MAP4 which became MPM- 2 immunoreactive following phosphorylation by the kinase(s) present in mitotic microtubule-depleted supernatant.
74 75
Figure 11 Figure 12. The effect of phosphatase inhibitors in the phosphorylation of interphase HeLa MAP4 by mitotic microtubule-depleted supernatant.
Phosphorylation of interphase HeLa MAP4 by mitotic microtubule-depleted supernatant was performed in buffer A in the presence or absence of 1 nM okadaic acid (OA) or 4 nM microcystin.
Panel A) Autoradiogram lanes 1,3,5, mitotic microtubule-depleted supernatant in buffer A (1) or buffer A + okadaic acid(3) or buffer A + microcystin(5). 2,4,6, mitotic microtubule-depleted supernatant + interphase boiled MAP4 in buffer A (2) or buffer A + okadaic acid(4) or buffer A + microcystin(6). Panel B) MPM-2 immunoblot lanes are same as panel A.
The arrowhead indicates interphase MAP4 which became MPM- 2 immunoreactive following phosphorylation by the kinase(s) present in mitotic microtubule-depleted supernatant.
76 77
Figure 12 Figure 13. Fractionation of mitotic HeLa microtubule- depleted supernatant kinase activities by ammonium sulfate.
Kinase assays were performed for each (NH4)2S04 fraction obtained from mitotic HeLa microtubule-depleted supernatant in the presence or absence of interphase HeLa MAP4 as an exogenous substrate. lanes 1, 0-25% (NH4)2S04 fraction of mitotic HeLa microtubule-depleted supernatant; 2, lane 1 + interphase MAP4; 3, 25-35% (NH4)2S04 fraction of mitotic HeLa microtubule-depleted supernatant; 4, lane 2 + interphase MAP4; 5, 35-45% (NH4)2S04 fraction of mitotic HeLa microtubule-depleted supernatant; 6, lane 5 + interphase MAP4; 7, 45-100% (NH4)2S04 fraction of mitotic HeLa microtubule-depleted supernatant; 8, lane 7 + interphase MAP4
78 79
MAP4
Figure 13
I Figure 14. Analysis of distribution of human Plk 1 in mitotic HeLa cells.
The distribution of human Plk 1 in the fractions obtained from the mitotic HeLa cell preparation including whole cell lysate, soluble cytoplasmic supernatant, microtubule-depleted supernatant, and microtubule pellet was analyzed by immunoblot analysis using R32 rabbit polyclonal anti-Plk 1 antiserum after 7.5 % SDS-PAGE.
lanes 1, mitotic HeLa whole cell lysate; 2, mitotic HeLa soluble cytoplasmic supernatant; 3, mitotic HeLa microtubule-depleted supernatant; 4, mitotic HeLa microtubule pellet
A
80 Figure 14 Figure 15. Immunoprecipitation of Plk 1 from mitotic HeLa cell.
Plk 1 was immunoprecipitated from mitotic HeLa cell soluble cytoplasmic supernatant using R32 serum as described (Golsteyn et al., 1995) (summarized in materials and method), and Western blot analysis was performed using monoclonal Plk 1 antibody to detect Plk 1 in immunoprecipitate.
lane 1, mitotic HeLa soluble cytoplasmic supernatant after removal of Plk 1 immunoprecipitate lane 2, Plk 1 immunoprecipitate
82 Figure 15 Figure 16. In vitro kinase assay of Plk 1 immunoprecipitate.
The Plk 1 immunoprecipitated from mitotic HeLa cell soluble cytoplasmic supernatant (see Figure 12) was used to perform kinase assays in vitro. Casein, interphase HeLa MAP4 or mitotic HeLa MAP4 were used as substrates for Plk 1. Assay condition was 20 mM HEPES (pH 7.4), 150 mM KC1, 10 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 5 mM NaF, 10
|JM ATP, 4 MCi [y-32P] ATP, and 0.5 mg/ml of dephosphorylated casein or 2 m ? of interphase or mitotic HeLa MAP4. lanes 1, Plk 1 immunoprecipitate (no exogenous substrate); 2, Plk 1 immunoprecipitate + casein; 3, Plk 1 immunoprecipitate + interphase MAP4; 4, Plk 1 immunoprecipitate + mitotic MAP4
84 85
MAP4
Figure 16 Figure 17. Time-course of phosphorylation of interphase HeLa MAP4 by Plk 1 immunoprecipitate.
The Plk 1 immunoprecipitated from mitotic HeLa soluble cytoplasmic supernatant (see Figure 12) was incubated with 2 (ig of interphase HeLa MAP4 for various periods of time (15 min, 3 0 min, 1 hr, 2hr) at 30 eC. The 7% SDS- PAGE was subjected, followed by autoradiography. The assay condition was same as in Figure 13.
The first lane (Endo) indicate the endogenous kinase activities of Plk 1 immunoprecipitate incubated for 15 min at 30 9C without added interphase HeLa MAP4.
86 87
MAP4
Figure 17 Figure 18. Immunoprecipitation of Plk 1 and MAP4 indicates they are not associated.
Plk 1 was immunoprecipitated as described in materials and methods from mitotic HeLa soluble cytoplasmic supernatant and Western bolt analysis was performed using polyclonal anti-MAP4 antibody. lanes 1, mitotic HeLa soluble cytoplasmic supernatant after removal of Plk 1 immunoprecipitate; 2, Plk 1 immunoprecipitate
88 MAP4 Figure 19. Peptide mapping of mitotic HeLa MAP4 by NCS cleavage.
Mitotic HeLa MAP4 was digested by N-chlorosuccinimide (NCS) (see materials and methods section) followed by 7% SDS-PAGE and MPM-2 immunoblot analysis.
lanes 1, control MAP4 which was not treated with NCS; 2, MAP4 fragments produced by NCS
90 210 kD 150-175kD
Figure 19 Figure 20. Peptide mapping of mitotic HeLa MAP4 by trypsin digestion.
Trypsin was added to the mitotic HeLa MAP4 (enzyme:substrate = 1:100) and incubated for various periods of time at 37 eC, followed by 4-12.5% gradient SDS-PAGE and Western blot analysis. The MAP4 digests were probed using two different antibodies: rabbit polyclonal anti-MAP4 and MPM-2.
Panel A) MAP4 immunoblot lanes 1, control (undigested MAP4); 2, 2 min digestion; 3, 5 min digestion; 4, 10 min digestion Panel B) MPM-2 immunoblot lanes are same as panel A.
92 Figure 20 Figure 21. Peptide mapping of mitotic HeLa MAP4 by chymotrypsin digestion.
Mitotic MAP4 was digested with chymotrypsin (enzyme:substrate = 1:100) for various periods of time at 37 9C. The MAP4 digests were run on 4-12.5% gradient SDS- PAGE followed by immunoblot analysis, using anti-MAP4 antibody or MPM-2.
Panel A) MAP4 immunoblot lanes 1, control (undigested MAP4); 2, 2 min digestion; 3, 5 min digestion; 4, 10 min digestion; 5, 20 min digestion Panel B) MPM-2 immunoblot lanes are same as panel A.
94 95
Figure 21 Figure 22. Peptide mapping of mitotic HeLa MAP4 by Staphylococcus aureus V8 protease.
Peptide mapping of mitotic HeLa MAP4 was performed using V8 protease. V8 protease was added to mitotic HeLa MAP4 (enzyme:substrate = 1:100) and incubated for various periods of time at 37 9C, followed by 4-12.5% SDS-PAGE and immunoblot analysis using anti-MAP4 polyclonal antibody or MPM-2.
Panel A) MAP4 immunoblot lanes 1, control (undigested MAP4); 2, 2 min digestion; 3, 5 min digestion; 4, 10 min digestion; 5, 20 min digestion Panel B) MPM-2 immunoblot lanes are same as panel A.
The arrowhead indicates the position of MAP4 peptide fragment that was cut out for sequence analysis (Panel A) .
96 97
B
1 2 3 4 5 1 2 3 4 5
—210 kD
-64 kD wm «m — 40 kD —30 kD
Figure 22 Figure 23. Microtubule binding assay of MAP4 peptide fragments generated by V8 protease digestion
Digestion of mitotic HeLa MAP4 was performed .as described in Figure 22. After protease digestion, MAP4 peptide fragments were incubated with purified rat brain tubulin under microtubule assembly conditions. The microtubule- binding MAP4 peptide fragments were separated from non binding fragments by centrifugation and both fractions were analyzed by MPM-2 immunoblot analysis. The same assay was carried out using undigested MAP4 as a control.
lanes 1, rat brain tubulin + undigested MAP4; 2, supernatant of lane 1 sample after microtubule binding assay; 3, pellet of lane 1 sample after microtubule binding assay; 4, rat brain tubulin + V8 protease digested MAP4; 5, supernatant of lane 4 sample after microtubule binding assay; 6, pellet of lane 4 sample after microtubule binding assay
98 99
1 2 3 4 5 6
-210 kD
—40 kD
Figure 23
I Figure 24. Peptide mapping of mitotic HeLa MAP4 by endoproteinase Arg-C.
Endoproteinase Arg-C was added to the mitotic HeLa MAP4 (enzyme:substrate = 1:50) and incubated for various periods of time at 37 9C, followed by 6-15% gradient SDS- PAGE and immunoblot analysis using MPM-2 antibody.
lanes 1, control (undigested MAP4); 2, 30 min digestion; 3, 1 hr digestion; 4, 2 hr digestion; 5, 18 hr digestion.
The arrowhead indicates the MAP4 peptide fragment that was cut out for sequence analysis.
100 210 kD
50 kD CHAPTER IV
DISCUSSION
The phosphoepitope specific monoclonal antibody MPM-2 (Davis et al., 1983) has been shown to recognize phosphoprotein components of mitotic cells that are associated with spindle microtubules and microtubule organizing structures (Vandre et al., 1984). One of the spindle associated MPM-2 reactive proteins has been identified as the microtubule associated protein MAP4 (Vandre et al., 1991). In addition to other MAPs (Vandre et al., 1991; Vandre et al., 1986), it has recently been shown that topoisomerase Iia and U p (Taagepera et al.,
1993), as well as the M-phase regulatory proteins cdc25, weel, mytl, and NIMA protein kinase (Kuang et al., 1994; Mueller et al., 1995a; Mueller et al, 1995b; Ye et al., 1995), are also MPM-2 reactive mitotic phosphoproteins.
The activated form of p42maPk has also been reported to be recognized by the MPM-2 antibody, however, the immunoreactive phosphorylated form of this protein is not restricted to mitosis (Taagepera et al., 1994). Thus, 102 103 phosphorylation of proteins at the MPM-2 epitope site could serve as an important posttranslational modification involved in the regulation of microtubule dynamics, cell cycle progression, and selected signal transduction events. Despite its potential importance, neither the MPM-2 phosphoepitope nor the MPM-2 epitope kinase have been clearly identified. MAP4 represents a relatively abundant MPM-2 reactive endogenous phosphoprotein that could serve as a model substrate to identify the MPM-2 epitope kinase and MPM-2 epitope site(s). We have examined the mitosis-specific phosphorylation of MAP4 in HeLa cells and the ability of exogenous and endogenous HeLa cell kinase activities to phosphorylate MAP4 in vitro, as well as analyzing the potential MPM-2 epitope site(s) of MAP4.
Microtubules were isolated from synchronized interphase and mitotic HeLa cells, but only the MAP4 associated with mitotic microtubules was found to be MPM-2 reactive. The mitotic form of MAP4 also exhibited slower electrophoretic mobility on polyacrylamide gels, which has been attributed to the hyperphosphorylation of the protein during mitosis. Our results have also demonstrated that MAP4 in mitotic cells incorporates labeled phosphate at a substantially greater rate than MAP4 in interphase cells, and this label is rapidly lost upon exit of cells from mitosis. Together with the MPM-2 immunoreactivity, the metabolic labeling offers direct evidence that MAP4 is phosphorylated in a 104 cell cycle dependent fashion, and hyperphosphorylated during mitosis in vivo.
Previous reports have indicated that MAP4 is an in vitro substrate for both p34cdc2 and MAP kinase (Ookata et al.,
1995; Hoshi et al., 1992). Comparison of the predicted amino acid sequence of MAP4 as determined for human, mouse, and bovine coding sequences (Chapin and Bulinski, 1991; West et al., 1991; Aizawa et al., 1990), revealed several conserved consensus phosphorylation sites, not only for p34cdc2 and MAP kinase, but also for casein kinase-2 (CK-2). Since the MPM-2 epitope on MAP4 is also expected to be conserved among these mammalian species, and these kinases have been implicated in the regulation of the cell cycle ( Mori et al., 1991; Aizawa et al., 1991; Hoshi et al., 1992; Tombes et al., 1991), we used these kinases to phosphorylate MAP4 in vitro to determine whether they were potential MPM-2 epitope kinases. Both mitotic and interphase HeLa MAP4 were phosphorylated by purified exogenous p44mpk and p34cdc2, but not by CK-2.
The 125 kD MAP ensconsin was a good substrate for CK-2, however. It is possible that the isolated HeLa MAP4 might contain endogenous phosphates at the CK-2 sites, and these might prevent the phosphorylation of the protein by this kinase in vitro. We have not examined this possibility by attempting to dephosphorylate MAP4 prior to the kinase assay, however. 105
Our results with exogenous kinases indicate that p34cdc2
has the capacity to convert interphase MAP4, which is not recognized by the MPM-2 antibody, into an MPM-2 reactive form. However, MPM-2 reactive MAP4 was only detected following extensive in vitro phosphorylation. This requirement for extensive phosphorylation is similar to that reported for the in vitro phosphorylation of MAP2 by cyclin dependent kinases, and the formation of hyperphosphorylated tau containing phosphoepitopes found in the Alzheimer's disease forms of the protein (Bauman et al., 1993). While tau was shown to be rapidly phosphorylated, the Alzheimer epitopes were not detected until after extensive periods of phosphorylation. It is possible that the MPM-2 site on MAP4 serves as a poor substrate for p34cdc2 kinase requiring extensive phosphorylation of the protein before it becomes detectable by immunoblot. Alternatively, the MPM-2 recognition site may involve multiple phosphorylation events. The slow conversion of interphase MAP4 into an
MPM-2 reactive form by exogenous p34cdc2 suggested the possibility that the endogenous HeLa MPM-2 epitope kinase was not p34cdc2 but might be a kinase related to p34cdc2.
These in vivo kinase might also be expected to be more efficient at phosphorylating the MPM-2 epitope site on MAP4. 106 Endogenous MAP4 kinase activities present in mitotic HeLa cells that might be related to p34cdc2 or MAP kinase were
found to be enriched in the microtubule pellet fraction, however, kinase activity also remained in the microtubule- depleted supernatant. Both MAP kinase and p34cdc2 kinase were found to be associated with the mitotic HeLa microtubules by immunoblot analysis. These microtubule- associated kinase activities phosphorylated a number of endogenous substrates associated with the microtubules including mitotic MAP4. Kinase assays were carried out using a number of different buffer conditions, and maximal MAP4 phosphorylation was found in buffers that favored p3 4 cdc2 or reiated kinase activities.
The MPM-2 epitope was generated on interphase MAP4 by both endogenous mitotic HeLa cell microtubule-associated kinases and kinase activity present in the microtubule depleted supernatant. The kinase activity in both of these endogenous samples rapidly converted MAP4 into an MPM-2 reactive form. Interestingly, formation of the MPM- 2 site on interphase MAP4 did not lead to a shift on the polyacrylamide gels to a higher apparent molecular weight typical of the endogenous mitotic MAP4. We were not able to recover the MPM-2 epitope kinase activity associated with the mitotic microtubule pellet following salt extraction. However, both the salt extract and the salt extracted microtubule pellet retained MAP4 kinase 107 activity. The MAP4 kinase activity present in the extracted microtubules remained despite the complete removal of endogenous MAP4 by the salt extraction. These results suggest that the MPM-2 epitope kinase was either inactivated during salt-extraction of the microtubules, or that multiple kinases, some of which are salt-extractable and some of which remain on the microtubules after salt- extraction, are required to generate the MPM-2 epitope. We have not succeeded in reconstituting the MPM-2 epitope kinase from the extracted microtubule preparation.
Mitosis-specific phosphorylation of proteins at the MPM-2 epitope has been implicated as an essential event in cell cycle progression. Identification of the MPM-2 epitope kinase(s) would be important for a more complete understanding of-the regulation of mitotic processes. Thus far, several reports have been published directed towards identifying the MPM-2 epitope kinase(s). Westendorf et al (1994) isolated cDNA encoding two MPM-2 reactive proteins and a variety of peptides encoding a potential MPM-2 epitope. They demonstrated that p34cdc2 immunoprecipitated from an M-phase cell extract could generate MPM-2 reactivity on some of these selected peptides. Kuang and Ashorn (1993) reported that MAP kinase and an unidentified high molecular weight protein kinase activity purified from unfertilized Xenopus eggs were capable of phosphorylating several immature Xenopus oocyte proteins on the MPM-2 epitope. Recently, Taagepera 108 et al (1994) showed that the activated form of p42mapk is an MPM-2 antigen, implying that MAP kinase kinase (MKK), also known as MEK, is an MPM-2 epitope kinase. Thus, three separate reports have implicated three different kinases as being MPM-2 epitope kinases. However, in each of these reports the MPM-2 substrates utilized or identified were not abundant endogenous MPM-2 reactive mitotic proteins. Although the identity of the endogenous HeLa MPM-2 epitope kinase has not been clearly established, the current studies suggest that it might be a cdc2-like kinase or related to the cdk family of kinases. Our results with MAP4 are consistent with previous reports implicating a role for cdc2 in generating the MPM-2 epitope (Mueller et al., 1995a; Ye et al., 1995) .
A recently described novel protein kinase family implicated in regulation of cell cycle processes is the human polo-like kinase (plk 1) (Golsteyn et al., 1995). It is a member of a newly emerging family of serine/threonine-specific protein kinases. The temporal activity and spatial localization of plk 1 suggest that it might be a candidate MPM-2 epitope kinase. Little is presently known regarding the phosphorylation site recognized by plk 1.
We examined the distribution of Plk 1 in the mitotic HeLa cell fractions generated during the isolation of HeLa 109 microtubules. We also examined whether Plk 1 could phosphorylate MAP4 in vitro. We found that Plk 1 was enriched in the mitotic microtubule pellet fraction. In addition, both interphase and mitotic MAP4 were good substrates for Plk 1 activity that was immunoprecipitated from the supernatant of a lysate prepared from mitotic HeLa cells. Interphase MAP4 was more heavily phosphorylated than the mitotic form of MAP4 by Plk 1. These results suggested that mitotic MAP4 substrate might contain endogenous phosphates occupying the Plk 1 phosphorylation sites that prevented further phosphorylation in vitro. The incorporation of phosphate into interphase HeLa MAP4 by Plk 1 increased up to 1 hr, however, interphase MAP4 was not converted to an MPM-2 reactive form after this phosphorylation. Thus, these results indicate that Plk 1 is a MAP4 kinase, however, it is not the in vivo MPM-2 epitope kinase. The identification and functional significance of Plk 1 phosphorylation of MAP4 will require additional studies.
Studies have also been carried out with other MPM-2 antigens in an attempt to elucidate the nature of the MPM- 2 epitope. Westendorf et al (1994) determined the sequences for MPM-2 binding on two cloned MPM-2 reactive proteins as LTPLK and FTPLQ. These two sequences partially overlap with the consensus phosphorylation sites of p34cdc2. Xenopus weel possesses several motifs for proline-directed kinases (Mueller et al., 1995a), and the protein contains two sites which fit the criteria for the phosphorylation by the MPM-2 epitope kinase proposed by Westendorf et al (1994). Taagepera et al (1994)
identified the site for the binding of MPM-2 in p42mapk as threonine-183, which is one of the essential residues phosphorylated during activation of the kinase. The sequence surrounding this site is FLTEYVA, and does not fit the consensus MPM-2 site proposed by Westendorf et al (1994), since it does not contain a proline residue C- terminal to the phosphorylated threonine. They also examined the effect of another nearby phosphorylated regulatory site in p42mapk, tyrosine-185, on the binding of MPM-2. They showed that the phosphorylation of tyrosine-185 was not required for MPM-2 recognition as this amino acid could be mutated to phenylalanine-185. The amino acid residue in this position was important for MPM-2 recognition, however, since a substitution with glutamic acid at position 185 greatly reduced the MPM-2 binding. Therefore, studies on the MPM-2 epitope suggest that it might either contain a proline-directed serine/threonine motif or a TEY-like sequence similar to that found in p42mapk.
Analysis of the human MAP4 sequence reveals the presence of 21 serine or threonine residues that are followed by a proline residue, providing several potential sites for proline-directed serine/threonine kinases. Indeed, human Ill
MAP4 contains 7 consensus sequences for p3 4cdc2 kinase,
and of these 2 are conserved in all MAP4 sequences available to date (see Table 2). In addition, TEY-like sites can be found in the N-terminal portion of the human MAP4 molecule, which are also conserved between the MAP4 from different species. These sites consist of the sequences TDY, TEF, and TNF with the threonine residue located at positions 45, 101, and 123 respectively. Since the MPM-2 epitope site of MAP4 has not been defined, either class of sites could be considered as potential MPM-2 epitope sites.
Peptide mapping of mitotic HeLa MAP4 was performed in order to examine the location of the MPM-2 epitope site on MAP4. A combination of chemical cleavage using NCS, and proteolytic digestions of mitotic MAP4 identified a 50 kD MAP4 fragment containing the MPM-2 epitope. The N-terminal amino acid residue of this peptide was located at position 449. This region corresponds to the proline-rich domain of MAP4, which contains the conserved phosphorylation sites for the proline-directed kinases. This result is consistent with our studies on the MPM-2 epitope kinase, which suggested that a cdc2-like kinase was a potential MPM-2 epitope kinase.
The MPM-2 site is expected to be sequence-specific but not conformation-specific, since other studies have shown that peptides of 15 amino acid residues can be recognized by MPM-2 using a phage display technique (Westendorf et al., 1994). We, therefore, expected small MAP4 phosphopeptides that retain MPM-2 reactivity could be generated following proteolysis. However, we were not able to obtain MPM-2 reactive MAP4 fragments smaller than 40 kD. A possible explanation for this result is that the MPM-2 epitope on MAP4 is more unique in that it requires a larger structural environment near the phosphorylation site for recognition, or that the phosphoepitope on smaller fragments of MAP4 were destroyed during the proteolytic digestion. Further studies should be carried out following purification of the 40-50 kD MPM-2 reactive MAP4 fragment generated by Arg-C. Further chemical cleavage using cyanogen bromide, which cuts at the C-terminal of methionine, could produce smaller fragments of the MPM-2 reactive MAP4 peptide, which would be appropriate for sequence analysis. Alternatively, further limited proteolytic digestion of the 40-50 kD MAP4 fragment using other enzymes could be performed to further define the MPM-2 epitope on the fragment.
While the results presented here have not defined the MAP4 MPM-2 epitope, they have shown that the epitope site is located in the proline-rich domain of MAP4. Thus, it appears unlikely that TEY-like sites located in the N- terminus of MAP4 are associated with the MPM-2 epitope on MAP4. The digestion results are consistent with our results showing that a cdc2-like kinase is capable of 113 generating the MPM-2 epitope, since the conserved proline- directed phosphorylation sites are located in the proline- rich domain. Further identification of MPM-2 reactive proteins in vivo and sequencing of the MAP4 phosphoepitope will be required to fully characterize the MPM-2 epitope, however.
It has been suggested that microtubule assembly promoting activity of MAPs is controlled by phosphorylation, and the involvement of specific kinases in a cell cycle dependent manner may explain how MAP phosphorylation may regulate microtubule dynamics in vivo. The involvement of
p34cdc2/CyCiin B complexes in regulating the dynamics of
mitotic microtubules has been demonstrated in cell-free extracts from Xenopus eggs (Verde et al, 1992; Ookata et
al., 1993). Addition of p34cdc2/cyclin B induced
shortening of microtubules nucleated by centrosomes that was attributed to an increase in the catastrophe frequency of the microtubules (Verde et al., 1992). Increased centrosomal nucleation of microtubules also occurred following phosphorylation of KCl-treated centrosomes by
the p34cdc2/cyclin B complex (Ookata et al., 1993). More
recently, the p34cdc2/cyclin B complex has been shown to
be associated with microtubules through the binding of cyclin B to the proline rich region of MAP4 (Ookata et al., 1995). In addition, the phosphorylation of MAP4 by p 3 4 c d2 c / C y C i i n b was reported to diminish the microtubule 114 stabilizing ability of MAP4. Thus, these results suggest that the direct interaction of MAP4 with p34cdc2/cyclin B may be important for the regulation of microtubule dynamics during mitosis by both targeting the M-phase kinase to its appropriate substrates in the cell and through direct phosphorylation of MAP4. Our results indicate that a p34cdc2-like kinase is capable of converting interphase MAP4 into a phosphorylated mitotic form recognized by the MPM-2 antibody. Thus, the MPM-2 phosphorylation site on MAP4 may be involved in the regulation of microtubule dynamics during mitosis. Defining the importance of this phosphorylation site in the function of MAP4 will require that it be fully characterized at the molecular level.
MAP4 appears to be a suitable endogenous substrate for the examination of mitosis-specific phosphorylation events including the role of cdc2-like kinases and plk 1 family members in the regulation of cytoskeletal dynamics and MAP4 function. In addition, MAP4 is an abundant MPM-2 reactive phosphoprotein whose characterization should lead to a better understanding of the MPM-2 epitope. The identification of the MPM-2 epitope on MAP4 coupled with the isolation of the HeLa cell MAP4 MPM-2 epitope kinase will be required for this analysis. Further, molecular analysis of MAP4 and other proteins that are MPM-2 reactive in vivo, as well as functional studies using 115 molecular techniques such as mutagenesis should address the significance of both the MPM-2 epitope and the kinase activity which is responsible for generating the MPM-2 epitope. The results presented here provide a foundation from which these studies can be developed. REFERENCES
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