And Integrin-Binding Protein CIB

376 Vol. 1, 376–384, March 2003 Molecular Cancer Research

The Serum-Inducible Protein Kinase Snk Is a G1 Phase Polo-Like Kinase That Is Inhibited by the Calcium- and Integrin-Binding Protein CIB

Sheng Ma,1 Mei-Ann Liu,1,2 Yi-Lu O. Yuan,1,3 and Raymond L. Erikson1

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA

Abstract cytokinesis. The Xenopus Plx1 activates Cdc2 indirectly by Identified as an immediate-early transcript, the serum- phosphorylation and activation of Cdc25C (2–4). Plx1 and inducible kinase Snk bears sequence homology with the Plk1 have been reported to be involve in nuclear translocation polo-like kinases. Endogenous Snk was detected in of cyclin B1 (5), but a recent report argued that phosphorylation

early G1 in NIH 3T3 cells, and nascent Snk showed a half- by Plk1 does not cause cyclin B1 to move into the nucleus (6). life of about 15 min. The kinase activity of endogenous Mitotic Plks also appear to contribute to centrosome matura-

Snk was detected in G1. Substitution of Thr-236 with a tion, disintegration of the Golgi apparatus in mitosis (7–9), glutamate residue increased Snk kinase activity by formation of bipolar spindles (3, 10, 11), regulation of the about 10-fold, whereas substitution of Lys-108 abolished anaphase-promoting complex (APC) (12–15), separation of the its kinase activity. Disrupting the polo-box did not sister chromatids (16), and completion of cytokinesis (17–21). significantly change Snk kinase activity. A GFP-C-Snk A role in regulating DNA replication was reported for the fusion protein showed polo-box-dependent localization budding yeast CDC5 (22). As cells exit mitosis, mitotic Plks are to the microtubule organizing center, and ectopic destroyed by ubiquitin-mediated proteolysis (15, 23, 24). expression of Snk in COS-7 cells induced changes in Because mitotic Plks are important regulators for mitosis, it is cell morphology, depending on Snk kinase activity and not surprising that their activity may be subjected to regulation the polo-box. The capacity of Snk to induce morpho- by the DNA replication checkpoint (24–26). The activity of logical changes was inhibited by the calcium- and mitotic Plks are also regulated by phosphorylation, and a polo- integrin-binding protein CIB. CIB co-immunoprecipitated like kinase kinase, xPlkk1, has been identified to phosphorylate with Snk and inhibited the kinase activity of Snk, and activate Plx1 (27). suggesting that CIB is a negative regulator for Snk In mammalian cells, two Plks, Fnk and Snk, were originally kinase activity. identified as immediate-early transcripts (28, 29). Further studies of Fnk show that the amount of Fnk increases from G1 to M phase, and Fnk is phosphorylated in mitosis, resulting Introduction in elevated kinase activity. Fnk is dephosphorylated at a later stage of mitosis and likely remains in the unphosphorylated The polo-like kinases (Plks) are a family of serine/threonine form when cells reenter G (23). Thus, it appears that Fnk may kinases closely related to polo of Drosophila melanogaster. 1 function during entry into the cell cycle as well as in mitosis. These kinases have been identified in evolutionarily diversified Prk (Plk3), which is the human homologue of Fnk, phosphory- eukaryotes, and they share a conserved NH -terminal kinase 2 lates Cdc25C in vitro, suggesting that it may play a role in entry domain and a homologous carboxyl terminus (1). A domain of into mitosis (30). Moreover, overexpression of Plk3 induced about 30 amino acid residues in the COOH terminus is highly apoptosis and incomplete cytokinesis, and a EGFP-Plk3 conserved among these kinases and thus dubbed the polo-box localizes to the cell midbody at the exit from mitosis (31). (1). The expression and activity of Plks are regulated in the Fnk may also play a role in cell adhesion (32). Genes encoding cell cycle. possible homologues of Fnk and Snk are also found in Many studies have been reported on the M phase Plks. Xenopus, and they are involved in oocyte maturation (33). In These enzymes are highly expressed and active during G -M of 2 Caenorhabditis elegans, three cDNA sequences were found to the cell cycle, and they are critical for events in mitosis and contain the polo-box (34, 35). One of them appears to be a homologue of mitotic Plks, whereas the functions of the other two remain unclear (36). In contrast to studies on the other Plks, the expression of Snk Received 10/31/02; revised 2/19/03; accepted 2/20/03. and its kinase activity in the cell cycle have not been reported. The costs of publication of this article were defrayed in part by the payment of One study reported that Snk, as well as Fnk, is expressed in rat page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. brain tissues, and their expression appears to be regulated by Grant support: NIH Grant CA42580 and GM59172 (R.L.E.). Note: R.L.E. is a John F. Drum American Cancer Society Research Professor. Requests for reprints: Sheng Ma, The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138. Phone: (617) 495-9686; Fax: (617) 495-0681. E-mail: [email protected] 2Present address: National Health Research Institutes, Taipei, Taiwan. Copyright D 2003 American Association for Cancer Research. 3Present address: Zycos, 44 Hartwell Avenue, Lexington, MA 02421.

neuronal activities (37). Our results reported here describe the restimulated to enter the cell cycle. Cell lysates were subjected expression and activity of Snk in cultured mammalian cells. We to immunoprecipitation and Western blot analysis with Snk also describe a functional interaction between Snk and the antisera. As shown in Fig. 1B, while Snk was not detected in calcium- and integrin-binding protein CIB. serum-starved cells, it was present in cells that were stimulated with serum for 1 h. The levels of Snk quickly decreased and Results were barely detectable at 2 h. In cells that were stimulated for 4, The Expression of Snk in the Cell Cycle 8, or 25 h, as well as those enriched for S or M phase The deduced amino acid sequence of Snk suggested that it population by treatment with hydroxyurea or nocodazole, no belongs to the family of Plks (29). Polyclonal Snk antisera were appreciable level of Snk was detected (Fig. 1B, lanes S and M) made to study the protein in the cell, and Fig. 1A shows that the Snk expression was also investigated in cells that reenter G1 antisera are highly specific for Snk and do not cross-react with after mitosis. NIH 3T3 cells arrested at M phase with either Plk1 or Plk3. To study Snk expression in the cell cycle, nocodazole were collected by mechanical shake-off and NIH 3T3 cells were synchronized by serum starvation and released into the cell cycle. Western blot analysis revealed that Snk started to appear 140 min after the release from the mitotic arrest, and the highest level was detected at 250 min (Fig. 1C) Only a small amount of Snk was still present 480 min after the release. On the other hand, the mitotic polo-like kinase, Plk, was most abundant in mitotic cells and nearly undetected by 70 min after the release. Cyclin B1, which is destructed at the end of mitosis, was completely degraded by 70 min after the mitotic release. These results indicate that Snk is expressed in early G1 during G0-G1 transition as well as in cycling cells. The brief expression of Snk in serum-stimulated cells suggested that Snk is turned over quickly. A pulse-chase experiment was carried out to determine the stability of endogenous Snk. Serum-starved NIH 3T3 cells were stimulated with serum in a methionine-free medium supplemented with [35S]methionine. The radiolabel was washed away with normal medium after 30 min, and cells were collected at various time points. Snk was immunoprecipitated from cell lysates using Snk antisera and analyzed by SDS-PAGE and autoradiography. The results showed that nascent Snk is rapidly degraded, with a half-life of about 15 min (Fig. 1D).

Analysis of the Kinase Activity of Snk The kinase activity of endogenous Snk was investigated in immunocomplex kinase assays. Snk was precipitated with Snk antisera from cells that had been stimulated with serum for 1 h, and immunocomplex kinase assay was carried out as described in ‘‘Materials and Methods.’’ The reaction mixtures were resolved by SDS-PAGE and analyzed by autoradiography. Casein was highly phosphorylated when incubated with the immunocomplex prepared from serum-stimulated cells (Fig. 2A). Phosphorylation of casein was not observed when serum-starved cells were used or when Snk antisera were FIGURE 1. A. The specificity of the Snk antisera. Western blot preincubated with the immunogenic peptide (Fig. 2A), analyses were performed with polyclonal antisera against Snk or Plk1, or anti-Plk3 monoclonal antibody as indicated. Lanes 1 – 3, COS-7 cell lysates suggesting that phosphorylation of casein is due to Snk kinase transfected with plasmids encoding HA-Plk1 (lane 2) or Flag-Snk (lane 3). activity. Phosphoamino acid analysis of phosphorylated casein Lane 4, Sf9 insect cell lysate containing ectopically expressed Plk3. Molecular size markers (in kilodaltons) are shown. B. Serum-starved NIH indicated that only serine and threonine residues were 3T3 cells were stimulated with 10% calf serum, and Snk was immunopre- phosphorylated (data not shown). cipitated and subjected to Western blot analysis with the Snk antisera. Because Snk has two highly conserved domains, including a S and M phase-enriched cell populations were obtained by treatment with hydroxyurea and nocodazole, respectively. C. NIH 3T3 cells were arrested serine/threonine kinase domain at the NH2 terminus and a polo- at M phase with nocodazole and collected by mechanical shake-off (Noc). box at the COOH terminus, point mutations were generated in Cells were then incubated in normal medium, and expression of Snk was these domains to study their function in Snk kinase activity. analyzed by Western blot analysis. D. Serum-starved NIH 3T3 cells were labeled in methionine-free medium containing 10% bovine calf serum and Thr-236, which aligns well with a putative activation 100 ACi/ml of Trans-35S label for 30 min (Pulse). After washing off phosphorylation site among serine/threonine kinases (38), was the radiolabel, cells were incubated in normal medium for the time indicated (Chase). Snk was immunoprecipitated and analyzed by SDS- substituted with several amino acid residues. The wild-type and PAGE. 35S-labeled Snk was detected by autoradiography. mutant Snk proteins were tagged with HA-epitope and

Plk has been reported to produce malignant transformation of NIH 3T3 cells, whereas overexpression of Plk3 resulted in incomplete cytokinesis and apoptosis (31, 39). To investigate Snk function in the cell, it was ectopically expressed in COS-7 cells, and confocal immunofluorescence microscopy was used to examine the cells expressing Snk. Cells growing on coverslips were transfected with plasmids encoding Flag-tagged Snk. After 24 h incubation, cells were fixed and those expressing Flag-Snk were detected with monoclonal anti-Flag antibody and labeled with FITC-conjugated secondary antibody. Snk was observed in the cytoplasm of cells. Moreover, cells expressing Snk showed an arborized morphology, with multiple processes extending from the cell body (compare Fig. 3A and B). Some Snk-expressing cells assumed a round morphology (Fig. 3C). Quantitation of the morphology of cells expressing Snk showed that cell arborization appeared to take place before their assuming the round morphology, as a cell population incubated 40 h after transfection showed an increased number of round cells (Table 1). Among the round cells, some had an intact nucleus, whereas a few showed chromosome condensation and appeared to be apoptotic (Fig. 3C). Expression of Snk mutants further showed that changes in cell morphology are specifically caused by an activity of Snk. The kinase activity is critical, in that the kinase-deficient FIGURE 2. The kinase activity of Snk. A. Snk was immunoprecipitated from NIH 3T3 cells that were serum starved or stimulated with serum for mutant, Snk.K108M, did not result in any significant changes, 1 h. Immunocomplexes were incubated with [g-32P]ATP, resolved by SDS- whereas Snk.T236E, which has an elevated kinase activity, PAGE, and analyzed by autoradiography. B. Thr-236 of Snk (WT) was caused cell arborization and rounding more rapidly (Table 1). changed to Glu, Asp, or Val (E, D, and V, respectively). HA-tagged proteins were produced in insect cells, and the kinase activity was However, Snk.fs, despite of having a kinase activity that is analyzed using immunocomplex kinase assays with anti-HA antibody. The comparable to that of the wild-type enzyme, showed a greatly top panel shows phosphorylated casein, and the bottom panel is a reduced capacity to induce morphological changes (Table 1), Western blot showing the amount of HA-Snk detected by Snk antisera. C. Flag-tagged Snk, a mutant with Lys-108 to Met substitution (KM), and a suggesting that the polo-box is also required. derivative with a disrupted polo-box (Snk.fs) were expressed in COS-7 A prior study showed that the polo-box of the mitotic Plk is cells. Immunocomplex kinase assays were performed with anti-Flag antibody. The amount of Snk was revealed by Western blot analysis with involved in its cellular localization and activity. Here we Snk antisera. Molecular size markers (in kilodaltons) are shown. examined COS-7 cells expressing a GFP-C-Snk, which carries an NH2-terminal GFP tag that swapped a peptide from Met-1 to Ala-309 in the kinase domain. Using a confocal fluorescence microscope, GFP-C-Snk was shown to localize primarily to an produced in insect cells. The kinase activities of these mutants area close to the nucleus in live unfixed cells (Fig. 4A). This were investigated by immunocomplex kinase assays with anti- area was determined to be the microtubule organizing center HA antibody. Results showed that although changing Thr-236 (MTOC) when cells were fixed and stained for a-tubulin to Asp or Val did not significantly affect Snk kinase activity, (Fig. 4D–F). The cellular localization of GFP-C-Snk appeared introduction of a negatively charged Glu caused about a 10-fold to be dependent on the microtubule network, in that treating the increase in Snk kinase activity (Fig. 2B). cells with cholchicine, which depolymerizes microtubules, Additional mutations were made to substitute Lys-108 in the putative ATP binding pocket or to disrupt the polo-box by introducing frameshift from Val-501 to Tyr-515. The resultant mutants, Snk.K108M and Snk.fs, respectively, were tagged with Flag-epitope and expressed in COS-7 cells. The kinase activity of these mutants was examined by immunocomplex kinase assays with anti-Flag antibody. Substitution of Lys-108 with methionine blocked Snk kinase activity, whereas Snk.fs was equally efficient as the wild-type in phosphorylating casein (Fig. 2C). Taken together, these results indicate that Snk is a typical serine/threonine kinase and that the kinase domain but FIGURE 3. Ectopic expression of Snk induces changes of cell not the polo-box is required for its kinase activity. morphology. COS-7 cells expressing Flag-Snk.K108M (A), which is a kinase-deficient mutant, or Flag-Snk (B, C) were stained with anti-Flag antibody and FITC-conjugated secondary antibody. Chromosomal DNA The Activity of Snk in Cultured Cells was stained with propidium iodide. A cell that contains condensed Among the three mammalian Plks, constitutive expression of chromosomes is indicated by an arrowhead. Scale bar, 25 Am.

Table 1. Quantitation of Morphologies of COS-7 Cells Expressing Snk and CIBa

24 h Post-Transfection 40 h Post-Transfection

Normal (%) Arborized (%) Round (%) Normal (%) Arborized (%) Round (%)

Vectorb 97.1 2.4 0.5 NDc ND ND Flag-Snk.KMd 90.2 1.5 8.3 ND ND ND Flag-Snk 26.1 F 1.0 37.9 F 2.9 36.0 F 3.8 18.7 F 9.0 24.8 F 4.2 56.5 F 5.0 Flag-Snk.TEe 14.0 F 11.3 33.8 F 6.5 52.2 F 4.4 21.2 F 9.2 24.8 F 9.4 53.9 F 16.4 Flag-Snk.fsf 78.0 16.0 6.0 83.5 8.5 8 HA-CIB 84.2 F 12.8 7.1 F 6.0 8.7 F 13.1 ND ND ND HA-CIB + Flag-Snk 91.5 F 6.6 4.2 F 3.6 4.3 F 2.9 66.2 F 12.6 20.7 F 12.2 13.1 F 0.9 HA-CIB + Flag-Snk.TE 85.1 F 1.8 5.7 F 5.1 9.3 F 6.2 68.2 F 5.6 9.4 F 5.9 22.4 F 2.9 aCells grown on coverslips were fixed with paraformaldehyde and permeabilized with cold methanol. Cell morphologies were scored independently by three individuals. Numbers shown are the average of either two or three counts. Please refer to the text for a description of the morphologies. bCells transfected with vector DNA were stained with anti-a-tubulin antibody and labeled with Texas Red. Cells from randomly chosen fields were scored for their morphology. cND represents not determined. dSnk.KM indicates the kinase-deficient mutant, Snk.K108M. eSnk.TE denotes the hyperactive mutant, Snk.T236E. fSnk.fs represents a Snk derivative with the polo-box disrupted by a frameshift mutation. resulted in dispersed GFP-C-Snk signal (Fig. 4B). In contrast, prepared after 27 h incubation. The lysates were subjected to the localization of GFP-C-Snk was not altered in cells treated immunoprecipitation using monoclonal antibody specific for with cytochalasin D, which disrupts the actin network (data not HA or Flag epitope, and the presence of Flag-Snk and HA-CIB shown). However, GFP-C-Snk.fs, which carries the frameshift in the immunocomplexes was detected by Western blot analysis mutation in the polo-box, showed a rather dispersed localization with anti-Flag and anti-HA antibody, respectively. Western blot in the cytoplasm (Fig. 4C), suggesting that the polo-box is analysis showed that HA-CIB was present in the immunocom- involved in cellular localization of Snk. plex precipitated by anti-Flag antibody, and Flag-Snk was present in the anti-HA immunocomplex (Fig. 6A). The results Functional Interaction of Snk With the Calcium- and suggest an association of the two proteins in COS-7 cells. One Integrin-Binding Protein CIB Our previous studies with yeast two-hybrid screening identified a Snk-interacting protein, which was deposited into the Genbank in 1996 (accession no. U83236). This protein was independently identified as the calcium- and integrin-binding protein (CIB) (40). Kauselmann et al. (37) subsequently reported the Snk and CIB interaction using yeast two-hybrid system and colocalization studies. Here we sought to test whether CIB has any effect on Snk activity in the cell. Plasmids encoding Flag-Snk and HA-CIB were co-transfected into COS-7 cells, and cell morphology was examined by immunofluorescence confocal microscopy. Cells expressing Flag-Snk were stained with FITC-conjugated antibody, while cells expressing HA-CIB were stained with Cy5-conjugated antibody. The results showed that HA-CIB was detected both in the nucleus and cytoplasm and that the expression of HA- CIB did not cause any apparent changes in cell morphology (Fig. 5, middle panel). However, whereas expression of Flag- Snk caused cell retraction and arborization, a cell that expressed both HA-CIB and Flag-Snk did not show significant morphological change (Fig. 5, indicated by an asterisk). That CIB was able to prevent Snk-induced morphological changes was further supported by scoring cells with various morphologies (Table 1). CIB also appeared effective in inhibiting the capacity of the constitutively active mutant, Snk.T236E, to FIGURE 4. Cellular localization of GFP-C-Snk. A – C. Images taken induce morphological changes (Table 1). from live unfixed COS-7 cells expressing GFP-C-Snk (A and B) or GFP- C-Snk.fs (C), which carries a frameshift mutation disrupting the polo-box. To investigate whether CIB and Snk interacted in COS-7 Cells were treated with colchicine in B. D – F. Images of cells that were cells, a co-immunoprecipitation assay was carried out. fixed and stained for a-tubulin. D. Cell expressing GFP-C-Snk. E. Microtubule network. F. Merged image of D and E, showing the Plasmids encoding HA-CIB and Flag-Snk were co-transfected localization of GFP-C-Snk to the microtubule organization center. Scale into COS-7 cells, and detergent-soluble cell lysates were bar, 25 Am.

shown to complement mitotic defects caused by CDC5 mutation in budding yeast (30, 43); however, our unpublished results show that Snk fails to efficiently complement the cdc5-1 temperature-sensitive defect in Saccharomyces cerevisiae (data not shown). This absence of detectable Snk expression and function in M phase strongly indicates that Snk is a member of the non-M-phase Plks. FIGURE 5. CIB inhibits morphological changes caused by ectopic In addition to the cell cycle regulation of Snk expression, expression of Snk. Plasmids encoding HA-CIB and Flag-Snk were co- endogenous Snk appears to be subjected to post-transcriptional transfected into COS-7 cells. Cells were fixed and permeabilized 24 h after transfection. HA-CIB and Flag-Snk were detected with anti-HA and anti- controls. Using pulse-chase analysis, nascent Snk was shown Flag antibody, respectively. Cells expressing Flag-Snk were stained with to have a half-life of about 15 min. The mitotic Plks such as FITC (left panel), and those expressing HA-CIB were labeled with Cy5 Cdc5 and Plk are substrates for the APC (12, 15, 44). (middle panel). A cell expressing both HA-CIB and Flag-Snk is indicated by an asterisk. Scale bar, 25 Am. Interestingly, a sequence of Snk from Arg-180 to Ser-188 is similar to that of the destruction box of cyclins (45), and a potential KEN box for Cdh1-APC is found in Snk from Lys- caveat is that CIB was reported to contain a myristoylation site at 149 to Asn-151 (46). Because the KEN box is shown to the NH2 terminus (41), which could be disrupted in HA-CIB. regulate proteolysis in G1 (47), it is possible that Snk is Myristoylation of CIB and its subsequent localization to degraded through this pathway. Moreover, a PEST sequence, the membrane may have an effect on its interaction with Snk which is present in many unstable proteins, is identified from in the cell. Arg-412 to Arg-426 in Snk using the PEST-FIND computer There are at least two scenarios whereby an association of program (48). It appears that as is the case for mitotic Plks, CIB can affect Snk activity in the cell. It is possible that CIB regulation of Snk levels may be an important mechanism for may interfere with the function of the polo-box, or CIB may modulating its activity in the cell. affect the kinase activity of Snk. To differentiate these two The kinase activity of endogenous Snk was demonstrated by possibilities, immunocomplex kinase assays were carried out phosphorylation of casein with immunocomplex kinase assays. to determine whether the interaction with CIB has any effect As expected, Snk phosphorylated serine and threonine residues on Snk kinase activity. Plasmids encoding Flag-Snk and HA- (data not shown). Studies of murine Plk and Xenopus Plx1 CIB were co-transfected into COS-7 cells. After incubation for 24 h, Flag-Snk was immunoprecipitated from cell lysates and assayed for its kinase activity. As shown in Fig. 6B, CIB diminished autophosphorylation of Snk and its phosphorylation of casein. The results suggest that CIB is an inhibitor of Snk kinase activity.

Discussion The serum-inducible kinase Snk was identified as an immediate-early transcript in NIH 3T3 cells, and its deduced amino acid sequence suggested that it is a member of the Plks (29). In this study, the expression of Snk in the cell cycle was examined. Endogenous Snk was detected in serum-starved NIH 3T3 cells that were stimulated with serum for 1 h. The amount of Snk rapidly decreased and became undetectable 4 h after stimulation. These observations are consistent with the expression profile of Snk message that was reported previously and suggest that Snk is expressed in G1 (29). In cells that were blocked at S or M phase, Snk was not detected. To examine whether Snk is expressed in cycling cells that reenter G1, NIH 3T3 cells were treated with nocodazole and subsequently released from the resultant mitotic arrest. Snk appeared 70 min FIGURE 6. Functional interaction between CIB and Snk. A. Co- immunoprecipitation of CIB and Snk. Detergent-soluble lysates of COS-7 after the complete degradation of cyclin B1, and because cells expressing HA-CIB and Flag-Snk were precipitated using anti-Flag destruction of cyclin B1 occurs at the end of mitosis (42), the (Flag IP). The presence of HA-CIB in the immunocomplex was detected data suggest that Snk is expressed in early G . The mitotic by a Western blot analysis using anti-HA (HA Blot). The membrane was 1 stripped and blotted with anti-Flag to reveal Flag-Snk (Flag Blot). The release experiment also showed that Plk is degraded before the immunoprecipitation was also performed with anti-HA (HA IP). Flag-Snk expression of Snk. In a similar experiment, the third member of was detected with anti-Flag (Flag Blot). A major cross-reactive band co- the murine Plks, Fnk, was shown to have altered electrophoretic migrated with HA-CIB (HA Blot). B. COS-7 cells were co-transfected with plasmids encoding Flag-Snk and HA-CIB. One ‘‘+’’ means 2 Ag of plasmid mobility when cells exit mitosis although its amount remained DNA was used for the transfection. Cell lysates were precipitated with anti- constant (23). An apparent overlapping of the expression of Fnk Flag antibody and immunocomplex kinase assays were performed. Phosphorylated proteins were visualized by autoradiography following with that of Plk and Snk raises a possibility of functional SDS-PAGE. The amount of Flag-Snk in the immunocomplex was shown redundancy among these Plks. Plk and Prk (Fnk) were both by Western blot analysis with anti-Flag antibody.

showed that an acidic substitution of a threonine residue in a Snk. Others have reported that ectopic expression of Plk3 putative activation loop of the kinase domain resulted in induced apoptosis (31), and it is possible that the apparent elevated kinase activity (3, 43). A similar substitution in Snk, apoptosis in cells expressing Snk is caused by phosphorylation where Thr-236 was changed to aspartate, caused about 10-fold of substrates that Snk and Plk3 have in common. increase in Snk kinase activity. Changing Lys-108 in the We originally identified the calcium- and integrin-binding putative ATP-binding domain to a methionine abolished the protein CIB as a Snk-interacting protein (Genbank accession kinase activity of Snk. It was reported previously that mutation no. U83236, 1996) using a yeast two-hybrid screening. in the polo-box did not affect Plk kinase activity (49), and this Subsequently, Kauselmann et al. (37) reported on the appears to be the case in Snk as well. Disruption of the most interaction of Snk and CIB and demonstrated their colocaliza- conserved region in the polo-box by a frameshift mutation tion in neuronal cells. In this study, we tested whether CIB has showed no significant effect on the kinase activity of Snk. any effect on Snk activity in the cell. Co-transfection of CIB These studies with Plk and Snk suggest that conserved with Snk diminished the capacity of Snk to induce morpho- mechanisms may be involved in regulating the activity of M logical changes. Because CIB and Snk co-immunoprecipitated and G1 Plks. from the cell lysate and that an immunocomplex kinase assay Functional analysis of Plk in previous studies suggested that revealed that co-expression of CIB with Snk decreased Snk both the kinase activity and the polo-box are required for in kinase activity, it appears that the association of CIB with Snk vivo mitotic functions of the enzyme (49). Lee et al. (49) may inhibit the kinase activity of Snk. Calmodulin, which showed that the polo-box is involved in cellular localization of shares sequence homologies with CIB, modulates activity of Plk. In this report, we showed that both the kinase activity and CaM-kinases through binding to the non-catalytic domain (51). the polo-box are required for Snk activity in the cell and that the It is also possible that CIB may inhibit Snk kinase activity by polo-box is involved in cellular localization of Snk. COS-7 recruiting a phosphatase, similar to the action of calcineurin B cells expressing Snk showed an arborized cell morphology, (40), although this appears unlikely, as CIB apparently inhibited with extended processes growing out of the cell body. the activity of a constitutively active mutant, Snk.T236E Expression of the kinase-deficient mutant of Snk did not cause (Table 1). Thus, CIB seems to be a direct modulator of Snk any significant morphological changes, nor did the expression kinase activity. Yeast two-hybrid interaction analysis showed of the polo-box frameshift mutant of Snk, although this mutant that CIB interacted strongly with the full-length Snk, whereas its has a kinase activity comparable to that of the wild type. On the interaction with the NH2-terminal kinase domain or the COOH- other hand, the mutant with an elevated kinase activity terminal domain, which retained an intact polo-box and other produced changes more rapidly. These data suggest that conserved sequences, was negligible (data not shown). Thus, it perhaps Snk contributes to morphological changes in the cell appears that CIB recognizes a structural feature in full-length as it exits mitosis. It is interesting that the expression of Snk in Snk. The mechanism of the interaction of CIB with Snk merits cells reentering G1 after mitotic arrest coincided with the further investigation, as CIB also showed interaction with Fnk flattening and spreading of round mitotic cells, which was (32) and could provide a phosphorylation-independent mecha- clearly visible under a light microscope (data not shown). It is nism for the regulation of the kinase activity of Plks. It should possible that Snk may be involved in the reorganization of the also be considered that CIB contains a myristoylation sequence cytoskeleton after mitosis. Support for this is the localization of and can be myristoylated and localize to the cell membrane (41), an NH2-terminally tagged GFP-C-Snk fusion protein to the which may affect its interaction with Snk and Fnk. MTOC in a polo-box-dependent fashion. Recently, Plk3 has Co-expression experiment showed that the inhibitory effect been reported to localize to the centrosome (50), and Sak, of CIB on Snk-induced morphological changes diminished over which shares homology with plks in the COOH terminus but time, as cells co-transfected with CIB and Snk showed an lacks a clearly defined polo-box, also showed localization to the increase in abnormal morphologies after incubation for 40 h. centrosome (51). Thus, the presence of the polo-box and This may be caused by changes in relative amount of Snk and unidentified sequence elements appears to predict subcellular CIB in the cell, or it is possible that the interaction between CIB localization of plks. It is noteworthy that in contrast to our and Snk is affected by other cellular factors. For example, CIB results, when a Snk-GFP fusion protein was used to study has two EF-hand motifs and binds Ca2+ (40), and previous cellular localization of Snk in COS-7 cells, no morphological studies have shown that Ca2+ is able to mediate the interaction changes were reported, and the fusion protein did not show of CIB with integrin (52). Therefore, it is possible that the specific localization to MTOC (37). It is possible that the GFP, interaction of CIB with Snk is affected by cellular Ca2+ which in this case was fused to the COOH terminus of Snk, concentration. This may be relevant to the role of Snk in may have disrupted the structure of the COOH terminus of Snk neurons, where CIB and Snk showed colocalization and have and the function of the polo-box. the potential to regulate neuronal activities (37). COS-7 cells expressing Snk for an extended time showed signs of apoptosis such as chromosome condensation. Expres- sion of a hyperactive mutant of Snk, Snk.T236E, caused more Materials and Methods cells to appear apoptotic 24 h after transfection. Given that Cell Culture and Chemicals endogenous Snk is present at low levels and quickly turned COS-7 and NIH 3T3 cells were obtained from American over in vivo, it appears that unregulated Snk activity is Type Culture Collection and maintained in DMEM supple- detrimental to the cell. Therefore, the apoptosis-like phenotype mented with 10% heat-inactivated fetal bovine serum (Intergen, induced by Snk may be an artifact of unregulated expression of Purchase, NY) or 10% heat-inactivated normal calf serum

(Intergen), respectively. Penicillin G (100 units/ml) and blot analysis using Snk antisera following protocols published streptomycin (100 Ag/ml) (Life Technologies, Inc., Rockville, previously (53). For immunocomplex kinase assays, RIPA MD) were included in the medium. Cells were incubated at buffer was substituted with Lysis buffer A [10 mM potassium 37jCin8%CO2. Serum starvation of NIH 3T3 cells was phosphate (pH 7.1), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, carried out by incubation in DMEM plus 0.5% calf serum for 2mM DTT, 2 mM Na3VO4,50mM h-glycerophosphate, 0.5% 30 h, and cells were stimulated with 10% calf serum. NIH 3T3 Triton X-100]. Anti-HA (12CA5, 2.8 Ag) or anti-Flag (M2, cells were treated with 1 mM hydroxyurea or 200 ng/ml of 5 Ag) antibodies were used to precipitate HA-Snk from Sf 9 and nocodazole for 18 h to obtain S or M phase-enriched cell Flag-Snk from COS-7 cells, respectively. The immunocom- population. To study Snk expression in cells that reenter the cell plexes were washed twice with Lysis buffer A, once with ST cycle, NIH 3T3 cells were treated with 200 ng/ml of [50 mM Tris-HCl (pH 7.2), 150 mM NaCl] containing 1 mM nocodazole for 18 h and collected by mechanical shake-off. DTT, and once with kinase buffer [25 mM Tris-HCl (pH 7.5), These cells were washed three times with cold DMEM plus 10 mM MgCl2,1mM DTT]. Kinase assays were performed by 10% calf serum and then incubated in pre-warmed growth incubating the immunocomplexes in 30 Al of kinase buffer medium. Colchicine treatment of COS-7 cells was carried out containing 5 ACi of [g-32P]ATP, 166 Ag/ml myelin basic protein for 2.5 h at 37jC at a final concentration of 40 Ag/ml. Transient (MBP) or casein, 0.1 mg/ml ovalbumin, and 50 AM ATP for transfection of COS-7 cells was performed using the Calcium 30 min at 30jC. Phosphate Transfection Kit (5 Prime-3 Prime Inc., Boulder, CO) or the GenePORTER transfection reagent (Gene Therapy Metabolical Labeling of Snk in NIH 3T3 Cells Systems, San Diego, CA), following the manufacturer’s To determine the half-life of endogenous Snk, pulse-chase protocols. Chemicals were purchased from Sigma, St. Louis, experiments were performed. Serum-starved NIH 3T3 cells MO unless stated otherwise. were washed with serum- and methionine-free DMEM medium (Life Technologies) and incubated in this medium for 30 min. Expression of Snk in Insect Cells and Mutagenesis of Snk Trans-35S label (ICN) and bovine calf serum were then added to HA-Snk was produced by fusing an NH2-terminal HA a final concentration of 100 ACi/ml and 10%, respectively. Cells epitope to the second amino acid residue of Snk by cloning a were grown at 37jC for 30 min before the radiolabel was SacI-EcoRI fragment of Clone 2 (29) into a baculovirus washed off with DMEM plus 10% serum. Cells were further transfer vector, pAC702 (Invitrogen), and expressed in Sf 9 incubated in DMEM containing 10% serum for the time cells using the BaculoGold Baculovirus Expression Vector indicated in the text. Cell lysates were prepared and subjected to System (PharMingen, San Diego, CA). Point mutations were immunoprecipitation with Snk antisera. The immunocomplexes generated in Snk by site-directed mutagenesis using a kit were resolved by SDS-PAGE, and 35S-labeled Snk was purchased from Amersham, Piscataway, NJ. Mutations were detected by autoradiography. confirmed by DNA sequencing. A frameshift mutation disrupting the polo-box was generated by cutting pSM227 at Ectopic Expression of Snk and Indirect a unique BstEII site just 5V to the polo-box, followed by blunt- Immunofluorescence Microscopy end ligation after S1 nuclease treatment. The COOH-terminal To ectopically express Snk in COS-7 cells, a Flag-epitope amino acid sequence was brought back into its original frame was fused to the Snk polypeptide that is encoded by a SacI- by cutting the resultant plasmid at a KpnI site in the polo-box, EcoRI fragment and deleted of the first methionine of Snk. The treating with S1 nuclease, and religation. resultant plasmid, pSM227, expresses the fusion protein from a cytomegalovirus (CMV) immediate-early promoter. The Immunoprecipitation, Western Blot Analysis, and kinase-deficient (Snk.K108M) and constitutively active Immunocomplex Kinase Assays (Snk.T236E) mutants were similarly expressed. For indirect Antisera were raised against an NH2-terminal peptide of immunofluorescence studies, COS-7 cells were seeded on 22 Â Snk, corresponding to amino acid residues Asp-29 to Gly-42, 22 mm acid-washed glass coverslips in a six-well tissue culture and the COOH terminus, from Ser-310 to Asn-682, that was plate and grown for 16–20 h. Cells were transfected with purified from insect cells as a GST fusion protein. The affinity- plasmid DNA and further incubated for the time indicated in the purified antisera were able to detect ectopically expressed Snk text. After two washes with PBS, cells were fixed at 4jCfor and various truncated Snk fragments with expected electro- 30 min in 4% paraformaldehyde-PBS (pH 7.4). Fixed cells phoretic mobility (data not shown). To detect endogenous Snk were washed three times with PBS and permeabilized with cold from NIH 3T3 cells, cells were lysed in 1 ml of radio- methanol for 2 min. The coverslips were then blocked with 6% immunoprecipitation assay (RIPA) buffer [10 mM Tris-HCl BSA for 30 min at room temperature. Primary antibodies were (pH 7.2), 150 mM NaCl, 1% sodium deoxycholic acid, 1% applied overnight at 4jC and secondary antibodies were applied Triton X-100, 0.1% SDS], and the lysates were clarified by for 30 min at 25jC. Monoclonal anti-Flag M2, anti-HA 12CA1, centrifugation at 100,000Âg for 30 min. Snk antisera (3–4 Ag) and rabbit polyclonal anti-HA (Santa Cruz Biotechnology, were added to the supernatant and incubated for 1 h at 4jC. Santa Cruz, CA) were diluted to a final concentration of 5 Ag/ml Protein A-conjugated with Sepharose 4B (Zymed, San in PBS containing 3% BSA; FITC-conjugated anti-mouse IgG Francisco, CA) was added and incubated for 30 min. (Boehringer Mannheim, Mannheim, Germany) and Texas Red- Precipitated immunocomplexes were washed three times with conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR) RIPA buffer, resolved by SDS-PAGE, and subjected to Western were diluted 1:200 in PBS containing 1% BSA. Propidium

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Downloaded from mcr.aacrjournals.org on September 28, 2021. © 2003 American Association for Cancer Research. The Serum-Inducible Protein Kinase Snk Is a G1 Phase Polo-Like Kinase That Is Inhibited by the Calcium- and Integrin-Binding Protein CIB1 1 NIH Grant CA42580 and GM59172 (R.L.E.). Note: R.L.E. is a John F. Drum American Cancer Society Research Professor.

Sheng Ma, Mei-Ann Liu, Yi-Lu O. Yuan, et al.

Mol Cancer Res 2003;1:376-384.

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