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Home , Cyclin, Hyperphosphorylation, Protein phosphatase, Protein phosphatase 1

Proc. Natl. Acad. Sci. USA Vol. 91, pp. 6408-6412, July 1994 Biochemistry and inactivation of 1 by -dependent (cel cyck/protein IhIsphor-ltln/i t / -dc mutagens) MARIAM DOHADWALA*, EDGAR F. DA CRUZ E SILVAt, FREDERICK L. HALLS, RICHARD T. WILLIAMS', DENISE A. CARBONARO-HALL0, ANGUS C. NAIRNt, PAUL GREENGARDt, AND NORBERT BERNDT*§ Departments of *Pediatrics and of *Surgery, Childrens Hospital Los Angeles, University of Southern California, Los Angeles, CA 90027; and tLaboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021 Contributed by Paul Greengard, March 18, 1994

ABSTRACT Protein phohatase 1 and protein phospha- for this site is suggested by the fact that it is also present in te 2A contain potential phosphorylation sites for cyclin- PP1 isoforms identified in Aspergillus (9), yeast (10), Dro- dependent kinases. In the present study we found that rabbit sophila (22), and plants (23), even though the C terminus is skeleal muscle 1, as well as recombinant the least conserved region of the molecule (7). Here, we protein phosphatse la and protein phosphatase 1yl, but not report that PPla and PPlyl can be phosphorylated and protein phosphatase 2A, was phosphorylated and inhibited by inactivated in vitro by cdks. In vitro expression and site- cdc2/ and cdc2/. Phosphopeptide mapping directed mutagenesis of PPla confirmed the cdk phosphor- and phospho analysis suggested that the phosphor- ylation to occur exclusively at Thr-320. Finally, we demon- ylation site was located at a C-terminal . Neither strate -dependent changes in PP1 activity, raising cdc2/cyclin A nor cdc2/cyclin B phosphorylated an active the possibility that this may be attributed in part to direct form of protein phosphatase la in which Thr-320 had been phosphorylation of PP1 by cdks. mutated to alanine, indicating that the phosphorylation oc- curred at this threonine residue. Furthermore, protein phos- MATERIALS AND METHODS phatase 1, but not protein phosphatase 2A, activity was found to change during the cell cycle of human MG-63 Chemicals. Phenylmethylsulfonyl fluoride was from cells. The observed oilio in protein phphatae 1 activ- Pierce. [y-32P]ATP was obtained from ICN. Chromatography ity during the cell cycle may be due, at least in part, to media and the Escherichia coli expression vector pDR540 phosphorylation of by cyclin-dependent were purchased from Pharmacia LKB. was kinass. Together, the results suggest a mcanis for direct obtained from LC Services (Woburn, MA). N-chlorosuccin- regulation of protein phosphatase 1 activity. imide (NCS) and all other chemicals were from Sigma unless otherwise stated. A family of /threonine-specific protein kinases termed . The catalytic subunits of PP1 and PP2A were cyclin-dependent protein kinases (cdks) plays a key role in purified from rabbit skeletal muscle to near homogeneity driving the cell cycle (1-4). Distinct perform different essentially as described by Cohen et al. (24), with the tasks in specific phases of the cell cycle by binding to and following modifications: The initial ion-exchange chromatog- activating different cdk catalytic subunits. Thus, cyclin B raphy was performed on DEAE-Sepharose fast flow, and the promotes the G2/M transition and cyclin A is required for the final gel filtration step was carried out on Sephacryl S-100 onset or maintenance of , while three G1 cyclins (C, HR. cdc2/cyclin A was purified as described (25, 26). D, and E) may regulate the cell's commitment to S phase (5). b, phosphorylase , and inhibitor 2 Clearly, the phosphorylation of key proteins is required for were kindly provided by Balwant S. Khatra (California State cell cycle progression to occur. Since the level of protein University, Long Beach). cdc2/cyclin B was obtained from phosphorylation depends on the ratio of and Steven L. Pelech (University of British Columbia, Vancou- the identification and ver). protein phosphatase (PP) activities, Expresion and Site-Directed MutagensofPPla. The Nar regulation of PPs opposing the actions of cdks are of con- 1-HindIl fragment ofrabbit PP1a cDNA (27) was subcloned siderable interest. into the BamHI site of pDR540 and transformed into E. coli Based on enzymatic and physicochemical properties, the DH5a. Cells were induced by the addition of isopropyl major mammalian serine/threonine-specific PPs are desig- ,&D-thiogalactopyranoside, and expressed PPla was purified nated PP1, PP2A, PP2B, and PP2C (6-8). A number of by chromatography on heparin-Sepharose and Sephacryl studies have indicated a potential role for PP1 and PP2A in S-100 HR. the regulation of cell growth. Analyses of fungal mutants Site-directed mutagenesis was initiated by annealing a revealed that PP1 and PP2A are essential for (9-12), mismatched oligonucleotide, 5'-GTTGCGGGGTGGC- and the observation that okadaic acid is a powerful tumor GATGGGTCGGCCTCC-3', to the coding strand ofthe PP1a promoter (13) and specific inhibitor ofPP1 and PP2A (14) has cDNA. The underlined codon (where C was the only mis- led to suggestions that these two function as tumor match) replaced the original Thr-320 by alanine. The reaction suppressors in mammalian cells (15, 16). was completed by using the "Transformer" site-directed All ofthe mammalian PP1 isoforms identified so far (17-20) mutagenesis kit (Clontech) according to the manufacturer's bear a Thr-Pro-Pro-Arg sequence motif near the C terminus, instructions. Mutant clones were selected by discriminatory corresponding to the preferred sequence for cdk phosphor- ylation, (Ser/Thr)-Pro-Xaa-(Lys/Arg) (21). A potential role Abbreviations: cdk, cyclin-dependent protein kinase; NCS, N-chlo- rosuccinimide; PP, protein phosphatase. The publication costs ofthis article were defrayed in part by page charge §To whom reprint requests should be addressed at: Childrens Hos- payment. This article must therefore be hereby marked "advertisement" pital Los Angeles, Box 83, 4650 Sunset Boulevard, Los Angeles, in accordance with 18 U.S.C. §1734 solely to indicate this fact. CA 90027. 6408 Downloaded by guest on September 25, 2021 Biochemistry: Dohadwala et aL Proc. Natl. Acad. Sci. USA 91 (1994) 6409 restriction mapping, since the desired mutation eliminated an time-dependent manner and was accompanied by an inhibi- Hph I site present in the parent plasmid, and then confirmed tion ofPP1 activity (Fig. 1). In parallel experiments, PP1 was by DNA sequencing., phosphorylated to 0.5 mol of per mol of protein PP Assays. The [32P]phosphorylase a was pre- within 30 miin (Fig. 1A), and its activity decreased to 50%o of pared by incubating phosphorylase b and phosphorylase the initial value (Fig. 1B). The inactivation of PP1 by cdc2/ kinase in a molar ratio of 200:1 in the presence of 0.2 mM cyclin A required the presence of both ATP and the kinase ['y-32P]ATP (28). PP activity was determined with 10.2 ,AM (Fig. 1B). P2P]phosphorylase a (1 mg/ml) in a total volume of30 p. (24). Purified, recombinant mammalian PP1 isoforms expressed To distinguish between PP1 and PP2A activities, the assays in the baculovirus system (unpublished work) were also used were carried out in the absence and presence of2 nM okadaic to investigate the phosphorylation of individual isoforms acid (29), which selectively inhibits PP2A. One unit of (Fig. 2). These data show (i) that PP1 also served as a activity is defined as the amount that catalyzes the release of substrate for cdc2/cyclin B, and (ii) that both PPla and 1 nmol of Pi from phosphorylase a per minute at 30'C. PP1y1 were phosphorylated to similar levels. When the Phosphorylatlon Assays, Phosphopeptide Mapping, and incubation was carried out in the presence of 20 p.M okadaic Pbospho Amino Acid Analysis. The purified catalytic subunit acid, thereby completely inhibiting PP1 activity, the phos- of PP1 or a synthetic C-terminal peptide of PPla (Gly-Arg- phorylation of PP1 was stimulated to a greater extent, sug- Pro-Ile-Thr-Pro-Pro-Arg-Asn-Ser-Ala-Lys-Ala-Lys-Lys) gesting that the catalytic subunit of PP1 is capable of auto- was incubated with purified cdk for up to 60 min in 50 mM (Fig. 2, lane 2 vs. lane 3). Tris HCI, pH 7.5/1 mM dithiothreitol/0.1 mM EDTA/100 mM NaCI/10 mM MgCl2/0.1 mM [y-32P]ATP (specific ra- A dioactivity 2 1000 cpm/pmol) and then analyzed as follows. When intact PP1 or PP2A was used as substrate, the samples were resolved by SDS/12% PAGE (30) and analyzed by autoradiography. When the peptide was used as substrate, the samples were spotted onto phosphocellulose paper, and their 32p content was determined (25). For phosphopeptide mapping, cdk-phosphorylated PP1 was resolved by SDS/ PAGE and digested with NCS within the excised gel slice (31). The resulting peptides were separated by SDS/15% PAGE, the gel was stained with silver and dried, and auto- radiography was performed. Phospho amino acid analysis was carried out as described (32).

... . Human MG-63 osteosarcoma cells (8 B i10 . In v. x 105) were plated in 80-cm2 flasks, cultured, and synchro- nized essentially as described (33). Asynchronous cells were cultured for 48 hr in the presence of reduced serum (0.5% fetal bovine serum) in order to obtain Go/GI samples. These cells were then grown in the presence of aphidicolin (2.5 ,pg/ml) for 24 hr to arrest them at the G1/S boundary. Cells 60(1 in S phase were obtained 4 hr after the release of the aphidicolin block by washing. Nocodazole (50 ng/ml) was used for 30 hr to synchronize cells at G2/M. The cellular > 400 synchrony was monitored and characterized by fluores- cence-activated cell sorter (FACS) analysis (34). At the appropriate times, cells were collected by scraping and washed in 5 ml of Hanks' balanced salt solution. The cells 00 were centrifuged for 10 min at 700 x g, suspended in 1 ml of ice-cold 50 mM Tris HCl, pH 7.5/0.1 mM dithiothreitol/0.1 r.{(1 - mM EDTA/0.1 mM EGTA/0.1 mM phenylmethylsulfonyl fluoride/1 mM benzamidine/150 mM NaCl with leupeptin at 5 pg/ml, and homogenized in a Dounce tissue grinder. The im llim nuclear fraction was collected by centrifugation for 15 min at 1500 x g and 40C. The resulting supernatant was taken as the FIG. 1. Phosphorylation and inactivation of PP1 by cdc2 kinase. cytosolic fraction. The nuclear fraction was resuspended in The purified catalytic subunitofrabbit skeletal muscle PP1 (PP1U, 780 0.3 ml of the above buffer and mixed with 33 pl of 4 M NaCl, ng, corresponding to .20 pmol) was incubated for various times at vortexed, and briefly sonicated. Protein concentrations were 30TC with U5 milliunits of purified cdc2/cyclin A in a total volume of 50 A4. (A) Time course of phosphorylation. The reaction was determined according to Bradford (35). stopped by the addition of 10 g1 of 1% bovine serum albumin and 40 A4 of 25% trichloroacetic acid. Precipitated and washed proteins RESULTS were analyzed by SDS/PAGE and autoradiography. Lane 1, kinase only; lane 2, PP1 only; lanes 3-5, kinase plus PP1 (10, 20, and 30 min Phosphorylatlon and Inactivation of PP1 Catalytic Subunit of incubation, respectively). The phosphorylation of PP1 at 30 min in Vitro. The presence ofa consensus phosphorylation site for represented a stoichiometry of0.5 mol/mol. (B) Effect ofphosphor- cdk in PP1 prompted us to investigate whether PP activity ylation on the activity of PP1. Incubation conditions were identical mightbe controlled by phosphorylation. Although PP2A does to those described in A, except that radiolabeled ATP was omitted. not contain a preferred cdk site as defined by Moreno and At the indicated times, aliquots ofthe reaction mixture were diluted 1:600 with ice-cold 50 mM Tris HCl, pH 7.0/1 mM dithiothreitol/ Nurse (21), it has three (Ser/Thr)-Pro motifs that could 0.1% bovine serum albumin. Phosphatase activity was determined potentially be phosphorylated by -directed kinases immediately. Control incubations lacking either cdc2/cyclin A or (2). However, only PP1 was phosphorylated following the ATP were carried out simultaneously. Data represent the mean incubation of rabbit skeletal muscle PP1 or PP2A with values of three independent experiments. *, PP1 plus kinase plus cdc2/cyclin A. The phosphorylation of PP1 proceeded in a ATP; A, PP1 plus kinase; o, PP1 plus ATP. Downloaded by guest on September 25, 2021 6410 Biochemistry: Dohadwala et al. Proc. Nad. Acad Sci. USA 91 (1994)

I 2 3 4 j performed (Fig. 3). When phosphorylated PP1 was subjected to phospho amino acid analysis, only phosphothreonine was 1 4 detected (Fig. 3A). of phosphorylated PP1 with NCS generated four fragments of 25.0, 23.8, 13.7, and 12.5 GEPEE, kDa, as detected by silver staining (Fig. 3B, lane 1). The bulk 4E,I ofradioactivity was associated with the smaller peptide (Fig. 3B, lane 2). The peptide pattern obtained with NCS treatment is consistent with preferential cleavage at Trp-206 and Trp- 216 rather than at Trp-149 (Fig. 3 B and C). Of the four FIG. 2. Phosphorylation of recombinant PP1 isoforms by cdc2/ threonine residues that occur within the 12.5-kDa C-terminal were with B and cyclin B. PP1 isoforms phosphorylated cdc2/cyclin in the sequence Thr-Pro-Pro-Arg, [y32P]ATP and analyzed by SDS/PAGE and autoradiography. Lane peptide, only Thr-320, 1, kinase only; lane 2, kinase plus PPla; lane 3, kinase plus PPlaplus corresponds to the cdk consensus phosphorylation site, 20 pM okadaic acid; lane 4, kinase plus PPlyl. The amount of (Ser/Thr)-Pro-Xaa-(Lys/Arg) (21). These data strongly sug- phosphate incorporated into PPla was increased -1.5-fold in the gested Thr-320 as the site that was phosphorylated by the presence of okadaic acid (compare lanes 2 and 3). cdks in vitro. This conclusion was supported by two addi- tional findings. First, cdc2/cyclin A was found to utilize a Loailzatlon ofthe cdk Phosphorylation Site. To localize the PP1 C-terminal peptide as a substrate. With 25, 50, and 100 phosphorylation site, PP1 was phosphorylated by cdk, and pM peptide the phosphate incorporation was 6.3, 11.4, and phospho amino acid analysis and peptide mapping were 19.8 pmol/30 min, rates that were well within the range reported earlier with a number of other synthetic peptide B substrates (25). In addition, the presence of the peptide kDa lowered substantially the amount of phosphate incorporated into PP1 (data not shown). Phosphorylation of Wild-Type and Mutant PPla. Both 37.4 wild-type PPla and the PPlaT320A mutant were expressed in E. coli and yielded soluble, active proteins that, like rabbit skeletal muscle PP1, were sensitive to inhibition by okadaic 25.0 _ acid and inhibitor 2 (data not shown). Wild-type PPla, but 23.8 not PPlaT320A, was phosphorylated by cdc2/cyclin A (Fig. 4). The lack ofphosphorylation ofPPlaT320A, coupled with the identification of a single major phosphopeptide by two- 1 3.7 --_ dimensional mapping (unpublished work) and the recovery of 12.5 -m_ phosphothreonine by phospho amino acid analysis, identifies A .4 P-Set Thr-320 as the only site in PP1 phosphorylated by cdk.

D?: -4 P-Ihr

() ...

.4 1-Ivr I -1 1() | C' NCS Cleavage Sites T323'PPR

-*------+ 23.8

25.0

13 .7 12.5 FIG. 3. Localization ofthe cdkphosphorylation site. (A) Phospho amino acid analysis of PP1. In vitro phosphorylated PP1 was sub- jected to phospho amino acid analysis as described (32). Positions of the standard phospho amino acids were established by ninhydrin staining and are indicated with arrows. (B) Phosphopeptide mapping ofPP1 after phosphorylation by cdc2/cyclin A in vitro. Purified PP1 was phosphorylated, separated by SDS/PAGE, excised from the gel, FIG. 4. Determination of the cdk phosphorylation site in PPla. and digested with NCS (31). The resulting peptides were resolved in Approximately 1 Aig of recombinant wild-type or mutant PPla was an SDS/15% polyacrylamide gel and detected by silver staining, and incubated with cdc2/cyclin A for 30 min as described in Materials the gel was dried and exposed to film for 24 hr. Lane 1, silver-stained and Methods. Lane 1, kinase only; lane 2, PPla only; lane 3, peptides derived from PP1 (gel); lane 2, phosphopeptides derived PPlaT320A only; lane 4, kinase plus PPlaT320A; lane 5, kinase plus from PP1 (autoradiograph). (C) PP1 peptides obtained by NCS PPla. Since the kinase preparation used in this experiment was cleavage. The top bar represents the full-length PP1 protein. The somewhat less pure than that employed in earlier experiments, it residues potentially cleaved by NCS (Trp-149, Trp-206, contained a number of other proteins that were phosphorylaed by and Trp-216) are indicated by the vertical arrows. The consensus the kinase (lane 1). These were virtually completely dephosphory- sequence Thr-Pro-Pro-Arg (TP32PPR) for phosphorylation by cdks, lated in the presence of the mutant PP1aT320A (lane 4), while in the located near the C terminus of PP1, is also indicated. A schematic presence of the same amount ofwild-type PPla a signiflcant fraction representation of the peptides recovered after NCS cleavage is ofphosphoproteins persisted (lane 5). This observation is consistent presented. The numbers correspond to the calculated polypeptide with phosphorylation of PP1 inhibiting its activity. The arrow indi- molecular mass in kilodaltons. cates the position of PP1. Downloaded by guest on September 25, 2021 Biochemistry: Dohadwala et al. Proc. Natl. Acad. Sci. USA 91 (1994) 6411 40 nuclear or chromatin-associated activity levels remained fairly constant in all four phases examined (Fig. 5 Lower). U Cysosol 0 10 Nucleus tkobo0. 30- DISCUSSION The results of the present study allow several conclusions. .I-- 20- on E First, the catalytic subunit of PP1 can be phosphorylated -tl threonine in vitro by both cyclin A and cyclin B kinase complexes. Second, in vitro phosphorylation of PP1 leads to 0. 10- a decrease in phosphatase activity. Third, cdk phosphoryla- 0. tion of PP1a occurs exclusively at Thr-320. Fourth, cdks also phosphorylate PPlyl, presumably at Thr-311, which corre- sponds to Thr-320 in PPla. It is interesting that a similar A GO/GI GI/S S G2/M consensus site for cdk phosphorylation is found in fungal (9, 4UAnl. 10), Drosophila (22), and plant (23) PP1 isoforms. Although -11 the specific functions of the various PP1 isoforms are un- r_0 * Cytosol known at present, PPla has been implicated in the control of 0 Nucleus , 30 cell proliferation (19), mitosis (9-12, 36), and phosphoryla- 1- tion of the (20). Furthermore, the ._ pattern of PP1 activity changes that are observed during the 20 cell cycle of somatic cells is consistent with the notion that may function as cell cycle regulators. Indeed, 0 -10 our data are consistent with a number of findings by other workers: (i) serum withdrawal leads to an increase in the 6- lo ribosomal protein S6 phosphatase activity of PP1 (37); (ii) the

CI levels and activity ofinhibitor 2 oscillate during the cell cycle, v exhibiting one peak during S phase and another peak during A GO/G I GC/S S G2/M M phase (38), possibly contributing to the cytosolic activity profile reported here; and (iii) PP1 translocates from the FIG. 5. PP1 and PP2A activities during the course ofthe cell cycle cytoplasm to the nucleus during G2 (39), possibly explaining of MG-63 cells. Cytosolic (filled bars) and nuclear (hatched bars) the high level of nuclear PP1 activity measured in G2/M (Fig. extracts were prepared from synchronized MG-63 cells and subse- 5 Upper). In contrast, PP1 has also been reported to show quently assayed for PP1 (Upper) and PP2A (Lower). "A" represents peaks of activity during and M phase in Xenopus asynchronous cells. The activity measured in "nuclear" extracts of M-phase cells represents PP associated with chromatin. The error egg extracts (40). However, that finding may not be directly bars represent the standard error of the mean (6 < n < 8). comparable with the data presented here, as in the early embryonic cell cycle of Xenopus there is virtually no G1 One problem that we encountered was the varying degree phase and associated growth controls. In addition to the ofphosphate incorporation. With highly purified cdc2/cyclin known mechanisms for regulating PP1 activity by transloca- A, the maximal stoichiometry was about 0.5 mol of phos- tion of the catalytic subunit or its interaction with inhibitory phate per mol of PPla, whereas less-pure cdc2/cyclin A subunits, our data provide evidence for the direct inhibition preparations and also highly purified cdc2/cyclin B resulted of PP1 by cdk phosphorylation. It should be possible to in a stoichiometry ofonly about 0.1-0.2 (even in the presence address the question of the biological role of PP1 phosphor- of okadaic acid). This suggested that efficient phosphoryla- ylation by cdks, by transfecting cDNA encoding the active, tion of PP1 may be dependent not only on the purity of the phosphorylation-resistant mutant PP1aT320A into mamma- kinase preparation but also on the type of cyclin involved. lian cells. PP1 and PP2A Activities During the Cell Cycle. Since PP1 In vitro phosphorylation of PP1 as well as PP2A has been and PP2A have been implicated in cell cycle regulation and previously observed, albeit on C-terminal residues, our observations suggest that PP1 may be directly inhibited by the oncogenic kinase pp60src (41, 42). In both cases, a by one or more members ofthe cdk family, we examined PP1 concomitant decrease in phosphatase activity was also re- and PP2A activity in extracts from synchronized MG-63 ported. Those data and the data reported here support the cells. The nature of the assay employed was such notion of the existence of a regulatory domain within the C that the absence or presence of 2 nM okadaic acid was used terminus of PP1. An important question is whether PP1 is to determine both PP1 and PP2A activities (29). Neither phosphorylated in vivo, and if so, which isoforms are regu- aphidicolin nor nocodazole, the drugs used to obtain cells lated by which cyclin/cdks. The observed differences in arrested at the G1/S and G2/M boundaries, had any direct phosphorylation of PP1 by cdc2/cyclin A and cdc2/cyclin B effect on PP1 or PP2A activity in vitro (data not shown). may represent yet another example of substrate targeting that Cytosolic PP1 activity was maximal (16.1 ± 2.8 units/mg) appears to reside in the cyclin rather than the cdk subunit during quiescence, dropped sharply to about one-fourth of (43). Possibly, PPla is a better substrate for cyclin C-, D-, or that activity (4.3 ± 1.3 units/mg) at the G1/S boundary, and E-dependent kinases than the enzymes used in the present then persisted at levels corresponding to about one-fifth of study. Alternatively, since a number of cdc2-like kinases maximum (3.6 ± 1.1 and 3.3 ± 0.7 units/mg, respectively) for have been identified by recombinant DNA technology, it is the remainder of the cell cycle (Fig. 5 Upper). A very similar possible that one of these may phosphorylate PP1 more pattern was seen with nuclear PP1 activity, except for efficiently, although there is virtually no information avail- mitosis. From high levels (33.6 ± 2.7 units/mg) in Go/GI, the able on the substrate specificity and cyclin association of nuclear PP1 activity decreased to 15.2 + 4.3 units/mg at the these proteins. A third possibility would be that tyrosine G1/S boundary, further decreasing (to 6.8 ± 1.3 units/mg) kinases and serine/threonine kinases controlled by cyclins during S phase. During M phase, where nuclei per se no [which also have been implicated in oncogenesis (44-47)] longer exist, a relatively high level of PP1 activity (30.7 + 3.6 may act synergistically to inhibit PP1 activity. units/mg) was associated with pelleted chromatin (Fig. 5 The mitosis-specific form of cdk, cdc2/cyclin B, has been Upper). In the case of PP2A, both the cytosolic and the reported to activate PP1 in vitro by phosphorylating inhibitor Downloaded by guest on September 25, 2021 6412 Biochemistry: Dohadwala et al. Proc. Nad. Acad Sci. USA 91 (1994) 2 (48). This observation appears to be in contrast with the 14. Bialojan, C. & Takai, A. (1988) Biochem. J. 256, 283-290. work presented here and previous findings discussed above. 15. Cohen, P. & Cohen, P. T. W. (1989) J. Biol. Chem. 264, 21435- 21438. However, these results are not contradictory, since PP1 16. Haystead, T. A. J., Sim, A. T. R., Carling, D. A., Honnor, R. C., appears to be required for the completion of mitosis in Tsukitani, Y., Cohen, P. & Hardie, D. G. (1989) Nature (London) Aspergillus (9), yeast (10-12), and Drosophila (36). If this 337, 78-81. also holds true for mammalian cells, activation of the inhib- 17. Berndt, N., Campbell, D. G., Caudwell, F. B., Cohen, P., da Cruz itor 2-bound PP1 by cdk could possibly provide the PP1 e Silva, E. F., da Cruz e Silva, 0. B. & Cohen, P. T. W. (1987) activity required for exit from mitosis. The inactivation of FEBS Lett. 223, 340-346. PP1 observed here occurs at or near the G1/S transition and, 18. Cohen, P. T. W. (1988) FEBS Lett. 232, 17-23. 19. Sasaki, K., Shima, H., Kitagawa, Y., Sugimura, T. & Nagao, M. provided that phosphorylation of the catalytic subunit con- (1990) Jpn. J. Cancer Res. 81, 1272-1280. tributes to this phenomenon, most likely involves a kinase 20. Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., other than cdc2/cyclin B. Kilburn, A. E., Lee, W.-H. & Elledge, S. J. (1993) Genes Dev. 7, One protein whose function is known to be mediated by 555-569. phosphorylation and believed to play a central role in main- 21. Moreno, S. & Nurse, P. (1990) Cell 61, 549-551. taining cells in G1 is the prototypical tumor suppressor, the 22. Dombradi, V., Axton, J. M., Brewis, N. D., da Cruz e Silva, E. F., retinoblastoma protein (RB). RB is largely dephosphorylated Alphey, L. & Cohen, P. T. W. (1990) Eur. J. Biochem. 194, 739-745. throughout G1 and becomes phosphorylated near the G1/S 23. Smith, R. D. & Walker, J. C. (1993) Plant Mol. Biol. 21, 307-316. boundary. It is thought that transition to S phase requires 24. Cohen, P., Alemany, S., Hemmings, B. A., Resink, T. J., Strilfors, inactivation of RB by and that the P. & Tung, L. (1988) Methods Enzymol. 159, 390-408. dephospho form of RB actively blocks cell cycle progression 25. Hall, F. L., Braun, R. K., Mihara, K., Fung, Y.-K. T., Berndt, N., (49, 50). RB is phosphorylated and inactivated by members Carbonaro-Hall, D. A. & Vulliet, P. R. (1991) J. Biol. Chem. 266, of the cdk family in vivo, most likely by cyclin A- and/or 17430-17440. -dependent kinases (26, 51,52). Furthermore, in vitro 26. Williams, R. T., Carbonaro-Hall, D. A. & Hall, F. L. (1992) On- cogene 7, 423-432. studies demonstrated that RB is dephosphorylated by an 27. Berndt, N. & Cohen, P. T. W. (1990) Eur. J. Biochem. 190, 291- inhibitor 2-sensitive phosphatase present in mitotic extracts 297. (53). Using the yeast two-hybrid system, Durfee et al. (20) 28. Shenolikar, S. & Ingebritsen, T. S. (1984) Methods Enzymol. 107, cloned a gene encoding a phosphatase, possibly an alterna- 102-129. tively spliced isoform of PP1, PPMa2, that can associate with 29. Cohen, P., Klumpp, S. & Schelling, D. L. (1989) FEBS Lett. 250, RB. These findings, coupled with the characteristic changes 596-600. 30. Laemmli, U. K. (1970) Nature (London) 227, 68-685. in PP1 activity observed during the cell cycle, suggest a 31. Lischwe, M. A. & Ochs, D. (1982) Anal. Biochem. 12, 453-457. potential role for PP1 at or after the M/G1 boundary, where 32. Nairn, A. C. & Greengard, P. (1987)J. Biol. Chem. 262,7273-7281. RB dephosphorylation takes place. 33. Carbonaro-Hall, D. A., Williams, R. T., Wu, L., Warburton, D., In summary, our data provide biochemical evidence for a Zeichner-David, M., MacDougall, M., Tolo, V. & Hall, F. L. (1993) direct inhibition ofPP1 activity by a cyclin-dependent protein Oncogene 8, 1649-1659. 34. Adams, R. L. P. (1990) Cell Culture for Biochemists (Elsevier, kinase, a mechanism which bypasses the known regulatory Amsterdam). subunits of PP1 and which may have important implications 35. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. for the regulation of the mammalian cell cycle, as well as 36. Axton, J. M., Dombradi, V., Cohen, P. T. W. & Glover, D. M. other cellular processes involving PP1. (1990) Cell 63, 33-46. 37. Olivier, A. R. & Thomas, G. (1990) J. Biol. Chem. 265, 22460- We wish to thank Julius Peters (Childrens Hospital Los Angeles) 22466. for performing oligonucleotide synthesis and Hsien-bin Huang (The 38. Brautigan, D. L., Sunwoo, J., Labb6, J.-C., Fernandez, A. & Rockefeller University) for the purification of recombinant PP1 Lamb, N. J. C. (1990) Nature (London) 344, 74-78. insect cells. We are 39. Fernandez, A., Brautigan, D. L. & Lamb, N. J. C. (1992) J. Cell isoforms from baculovirus-infected Sf9 grateful Biol. 116, 1421-1430. to Balwant S. Khatra for his gift of purified phosphorylase b, 40. Walker, D. H., DePaoli-Roach, A. A. & Mailer, J. L. (1992) Mol. , and inhibitor 2 and to Steven L. Pelech for his Biol. Cell 3, 687-698. gift ofcdc2/cyclin B. This work was supported in part by U.S. Public 41. Johansen, J. W. & Ingebritsen, T. S. (1986) Proc. Nad. Acad. Sci. 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