Characterization, Subcellular Distribution, and Cell Cycle ... - CiteSeerX

Vol. 10, 611– 620, September 1999

Cell Growth & Differentiation

PCTAIRE-1: Characterization, Subcellular Distribution, and Cell Cycle-dependent Kinase Activity Sophie Charrasse,1 Ilaria Carena, Joerg Hagmann, Kathie Woods-Cook, and Stefano Ferrari2 Department of Oncology, Novartis Pharma AG, 4002 Basel, Switzerland [S. C., I. C., K. W-C., S. F.], and Friedrich Miescher Institute, 4057 Basel, Switzerland [J. H.]

Abstract PCTAIRE-1 is a member of the cyclin-dependent kinase (cdk) family whose function is unknown. We examined the pattern of PCTAIRE-1 protein expression in a number of normal and transformed cell lines of various origins and found that the kinase is ubiquitous. Indirect immunofluorescence indicated that PCTAIRE-1 exhibits cytoplasmic distribution throughout the cell cycle. Confocal microscopy showed that PCTAIRE-1 does not colocalize with components of the cytoskeleton or with the endoplasmic reticulum. We found that endogenous PCTAIRE-1 and ectopically expressed PCTAIRE-1 display kinase activity when myelin basic protein is used as an acceptor substrate. Similar to other members of the cyclin-dependent kinase family, PCTAIRE-1 seems to require binding to a regulatory subunit to display kinase activity. PCTAIRE1 activity is cell cycle dependent and displays a peak in the S and G2 phases. We show that the low level of kinase activity observed until the onset of S phase correlates with elevated tyrosine phosphorylation of the molecule.

Introduction Treatment of quiescent cells with growth factors or hormones induces reentry into the cell cycle. This is accompanied by an increase in the synthesis of RNA and protein and culminates in a doubling of protein mass before DNA synthesis and cell division (reviewed in Refs. 1 and 2). Crucial to a correct transition through the cell cycle is the timely activation of a subfamily of protein kinases, the cdks.3 The prototype of this family is encoded by the cdc2 gene, whose human homologue was identified by its ability to functionally

Received 3/8/99; revised 6/3/99; accepted 7/7/99. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 S. C. is a recipient of a fellowship from the Association pour la Recherche contre le Cancer, France. 2 To whom requests for reprints should be addressed, at Department of Oncology, K-125.4.01, Novartis Pharma AG, Klybeckstrasse 141, 4057 Basel, Switzerland. Phone: 41-61-696-1715; Fax: 41-61-696-3835; Email: [email protected]. 3 The abbreviations used are: cdk, cyclin-dependent kinase; HA, hemagglutinin; MBP, myelin basic protein; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; PVDF, polyvinylidene difluoride; BrdUrd, bromodeoxyuridine.

complement the yeast Schizosaccharomyces pombe p34cdc2 mutant (3). p34cdc2 activity is controlled by a network of activating and inhibitory signals, including protein-protein interactions, posttranslational modifications, and subcellular localization (4). The regulatory inputs converging on p34cdc2 are both intrinsic and extrinsic to the cell. Among the former is phosphorylation, which is responsible for timing and coordinating cell cycle events. Extrinsic signals are provided by the environment and target principally the levels of cdk inhibitors as well as G1 cyclins. A combination of both signals results in stimulation or inhibition of progression through the cell cycle (5). A number of kinases have been identified in mammalian tissues based on structural homology with p34cdc2 (6). The majority of these molecules have no counterpart in yeast. P34cdc2-related kinases play a variety of roles during the transition through the cell division cycle. These include monitoring progression through checkpoints (7–10) and regulating the activity of other cdk family members as well as that of RNA polymerase II (11, 12). Despite the significant progress in the knowledge of p34cdc2-related kinase function, understanding their mechanism of action at the molecular level awaits the identification of specific targets. A restricted number of physiological substrates have been identified thus far, such as the retinoblastoma protein Rb (13), the transcription factor E2F (14 –16), A- and B-Myb (17, 18), and the motor protein Eg5 (19). However, the majority of events triggered by p34cdc2-like kinases are still largely unknown. The ability to bind cyclins is a distinctive feature of p34cdc2-related kinases. Cyclins are sequentially synthesized during transition through the cell cycle and trigger the process of kinase activation upon binding to cdks (4). Not all cdks, however, have a known cyclin partner. These orphan cdks display a peculiar pattern of expression with high levels in postmitotic tissue (6). This suggests that their function is not restricted to the regulation of proliferation, and they may also control the state of differentiation (6, 20 –22). PCTAIRE-1 is one such member of the cdk subfamily. The kinase derives its name from the presence of a cysteine-for-serine substitution in the cyclin binding sequence PSTAIRE. Three distinct PCTAIRE isoforms have been identified in humans (6), two of which are also present in rodents (23, 24), and one in Dictyostelium (25). The gene for PCT-1 has been mapped to human chromosome Xp, close to the ubiquitin-activating enzyme E1 gene (23). The protein encoded by the PCT-1 gene displays a relative Mr of 55,000 and contains conserved motifs of the Ser/Thr protein kinase family (6). A 161-amino acid NH2-terminal extension and a 40-amino acid COOHterminal extension flank the central catalytic domain, which displays a 52% identity to p34cdc2 (6). In addition to the lack of cyclin binding, interaction of PCTAIRE-1 with cdk inhibitors has not been reported to date. Whether the NH2- and/or COOH-terminal extensions in PCTAIRE-1 may substitute for

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Fig. 1. Detection of the endogenous and ectopically expressed PCTAIRE-1 protein. A, protein extracts (15 mg) from various cell lines were examined by Western blotting using an antibody specific to PCTAIRE-1. Jurkat cells, Lane 1; COS-7 cells, Lane 2; Hs68 cells, Lane 3; NIH3T3 cells, Lane 4; human lung carcinoma A549 cells, Lane 5; human cervical carcinoma HeLa cells, Lane 6; human mammary carcinoma MCF7 cells, Lane 7; human gastric carcinoma GTL16 cells, Lane 8; human colon adenocarcinoma Colo 205 cells, Lane 9; 293 human embryo kidney cells, Lane 10; human epidermoid carcinoma A431 cells, Lane 11; lymph node prostate cancer LN-cap cells, Lane 12; human osteosarcoma Saos-2 cells, Lane 13; human glioblastoma U87 cells, Lane 14. B, extracts (10 mg) of wild-type (Lane 1) and SV40-transformed (Lane 2) WI38 fibroblasts were examined by Western blot analysis as described above. C, Western blot of GTL16 cell extract (15 mg). The membrane was probed with PCTAIRE-1 antibody (Lane 1) or with the same antibody preincubated with a 10-fold (w/w) excess of antigenic peptide (Lane 2). D and E, COS-7 cells ectopically expressing HA-tagged PCTAIRE-1 were extracted at 24 (Lane 3) or 36 h (Lane 4) after transfection. Controls include nontransfected cells (Lane 1) and cells transfected with empty vector (pcDNA3-HA; Lane 2). PCTAIRE-1 protein was revealed with the anti-PCTAIRE-1 antibody (D) or with the anti-HA 12CA5 antibody (E). The band at Mr 45,000 in E is nonspecifically detected by the antibody used to probe the membrane.

the effect of regulatory proteins on the activity of the kinase is still unknown. Peculiar to PCTAIRE-1 is the ability to bind calpactin I light chain and the 14-3-3 proteins, although no effect on kinase activity has been reported upon such interaction (26, 27). In the attempt to shed light on the role of PCTAIRE-1 during transition through the cell cycle, we characterized the expression pattern and kinase properties as well as the subcellular localization of the kinase. We found that PCTAIRE-1 is present in the cytoplasm throughout the cell cycle and displays kinase activity during S phase and the G2 phase. The peak of kinase activity correlated with dephosphorylation of tyrosine residues.

Results Pattern of PCTAIRE-1 Protein Expression. Northern blot analysis carried out on a number of tissues and cell lines indicated the presence of multiple transcripts for PCTAIRE-1 (6, 23). To test whether this may result in the translation of different isoforms of the kinase, we examined the pattern of PCTAIRE-1 protein expression in cultured cells of various origins. To this end, we used an affinity-purified polyclonal antibody directed to a COOH-terminal epitope in PCTAIRE-1. As shown in Fig. 1A, a band displaying an apparent Mr of 55,000 corresponding to the predicted size for PCTAIRE-1 was detected in all cell lines examined. When protein expression was compared in normal and SV40-transformed WI38 fibroblasts, PCTAIRE-1 levels were higher in

the transformed cells (Fig. 1B). A larger polypeptide was apparently present in some of the cell lines (Fig. 1, A and B). Longer exposure times revealed that the higher molecular weight band was detectable in all cell lines, with the exception of Hs68 and 293 cells. The presence of a larger PCTAIRE-1 isoform has been described previously in a study in which PCTAIRE-1 cDNA was translated in vitro (23) and may represent a product of differential splicing. To test whether this was the case, we performed a competition assay using the peptide antigen used to raise the antiserum. A 10-fold (w/w) excess of peptide resulted in ablation of both signals, indicating that the larger protein was immunologically related to PCTAIRE-1 (Fig. 1C). However, transient transfection of COS-7 cells with HA-tagged PCTAIRE-1 resulted in the expression of a single form of the kinase, which migrates with an apparent molecular weight that is slightly higher than the endogenous Mr 55,000 protein (Fig. 1, D and E, Lanes 3 and 4). This result, together with the data obtained from Western blot analysis, indicates that in most cells, a single form of the kinase is predominantly expressed. PCTAIRE-1 Kinase Activity. The catalytic domain of PCTAIRE-1 contains all of the conserved features of the Ser/Thr protein kinase family. To test whether PCTAIRE-1 displays phosphotransferase activity, we immunoprecipitated HA-tagged PCTAIRE-1 and used MBP as an acceptor substrate. HA-tagged PCTAIRE-1 appeared to efficiently phosphorylate MBP (Fig. 2D, Lane 3). Next we expressed

Cell Growth & Differentiation

Fig. 2. Purification of recombinant GST-PCTAIRE-1 and detection of PCTAIRE-1 kinase activity. Full-length PCTAIRE-1 was expressed as a GST-fusion protein in insect Sf-9 cells. The recombinant protein was affinity-purified on a glutathione-Sepharose 4B column as described in “Materials and Methods.” Purified GST-PCTAIRE-1 (2 mg) was resolved on a 10% polyacrylamide gel in the presence of SDS and either stained with Coomassie Blue (A) or analyzed by Western blotting with an antibody to PCTAIRE-1 (B) or GST (C). D, HA-tagged PCTAIRE-1 was ectopically expressed in HeLa cells, and kinase activity was determined using MBP as a substrate. Extracts from cells transfected with the empty vector (Lanes 1 and 2) or HA-tagged PCTAIRE-1 (Lane 3) were left untreated (Lane 1) or were immunoprecipitated with the 12CA5 antibody (Lanes 2 and 3). Samples were treated with protein G-Sepharose, and immunocomplexes were tested for kinase activity as described in “Materials and Methods.” Proteins were resolved by SDS-PAGE followed by autoradiography. E, in gel kinase assay. Equal amounts of protein (200 mg) derived from the extracts of untreated and H2O2-treated Jurkat cells (Lanes 1 and 2) or HA-tagged PCTAIRE-1-expressing Hs68 (Lane 3), HeLa (Lane 4), and COS-7 cells (Lane 5) were immunoprecipitated with anti-ERK2 (Lanes 1 and 2) or anti-PCT-1 antibody (Lanes 3–5). An asterisk indicates the position of ERK2. Lane 6 contains affinity-purified GST-PCTAIRE-1 (15 mg). Proteins were separated on a 10% polyacrylamide gel containing 0.5 mg/ml MBP under denaturing conditions. Renaturation of the resolved proteins and determination of kinase activity were carried out as described in “Materials and Methods.” F, purified GST-PCTAIRE-1 (5 mg) was examined for kinase activity on MBP before (Lane 1) or upon (Lane 2) a 2-h incubation on ice with 50 mg of fibroblast cell extract.

PCTAIRE-1 as a GST-fusion protein in insect cells and purified it to near homogeneity (Fig. 2, A–C). The purified protein was found to be devoid of kinase activity when tested on MBP (Fig. 2F, Lane 1) but could be fully activated upon incubation with fibroblast cell extract (Fig. 2F, Lane 2). This suggests that PCTAIRE-1 may need binding to a regulatory partner and/or posttranslational modification to display activity. To test this hypothesis, we performed an in situ kinase assay using immunopurified HA-tagged PCTAIRE-1 and MBP as an acceptor substrate (Fig. 2E). ERK2 was used as representative of kinases requiring only posttranslational modification to display activity. Whereas immunopurified ERK2 displayed kinase activity (Fig. 2E, Lane 2, asterisk), phosphorylation of MBP by HA-tagged PCTAIRE-1 did not occur under these conditions (Fig. 2E, Lanes 3–5). However, at this point we cannot rule out that, unlike ERK2, PCTAIRE-1 may fail to renature in this type of assay. The data above do not rule out the possibility that another kinase may associate with PCTAIRE-1 and be responsible for the phosphorylation of MBP. To address this issue, we generated two independent kinase-dead mutants by point mutating key residues involved with the catalytic process of

phosphorylation. HA-tagged D304N and K194A mutants were ectopically expressed in HeLa cells (Fig. 3A), immunoprecipitated, and assayed for kinase activity. The results presented in Fig. 3B show that both point mutants were devoid of kinase activity, thus ruling out the possibility that MBP phosphorylation by the wild-type kinase is due to contaminant, coprecipitating kinases. The possible interaction with cyclins was examined by a variety of means. First, pull-down studies were performed on the incubation of affinity-purified GST-PCTAIRE-1 with extracts of exponentially growing Hs68 cells. The result obtained showed no interaction between PCTAIRE-1 kinase and cyclin D1, E, A, B1, or B2 (data not shown). Second, in vivo studies were performed by immunoprecipitating ectopically expressed HA-tagged PCTAIRE-1 from HeLa cells or endogenous PCTAIRE-1 from Hs68 fibroblasts synchronized in S phase (see below). Western blot analysis carried out with an antibody to cyclin D1, E, A, G, and F was negative. Finally, in vivo labeling of transfected HeLa cells with [35S]methionine/[35S]cysteine followed by immunoprecipitation of HAtagged PCTAIRE-1 showed that no protein was bound to the kinase in a stoichiometric amount (data not shown).

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Fig. 3. Characterization of PCTAIRE-1 kinase-dead mutants. A, Western blot analysis of HA-tagged PCTAIRE-1 wild-type (Lane 2) and D304N (Lane 3) and K194A (Lane 4) point mutants upon ectopic expression in HeLa cells. HeLa cells transfected with empty vector (pcDNA3-HA) were used as a control (Lane 1). B, wild-type or kinase-dead HA-tagged PCTAIRE-1 mutants were immunoprecipitated from HeLa cell extracts, and kinase activity was determined as described in “Materials and Methods.”

Members of the cdk family display a typical pattern of activation during the cell division cycle, with their activity depending upon posttranslational modifications and proteinprotein interactions. We examined whether PCTAIRE-1 activity is also cell cycle dependent. To this end, we used Hs68, a human fibroblast cell line that can be easily synchronized by serum deprivation and is well characterized (28). Cells were starved by serum deprivation for 48 h and restimulated to enter the cell cycle by the addition of 10% FCS. Fig. 4A shows the extent of Hs68 synchronization and the orderly progression through the cell cycle. PCTAIRE-1 consistently displayed low kinase activity in G1 and a significant decrease at the G1-S-phase transition (Fig. 4B). Upon entry into S phase, PCTAIRE-1 activity increased rapidly, displaying a peak during the transition through the S phase and G2 phase. The overall level of endogenous PCTAIRE-1 protein did not change during progression through the cell cycle (Fig. 4C). To exclude cell type-specific effects or artifacts due to the method of synchronization used, we examined PCTAIRE-1 kinase activity in a different system. HeLa cells were arrested by double thymidine block, and the extent of synchronization upon release from the block was examined by flow cytometric analysis of DNA content (Fig. 5A). The high synchronicity of the system was confirmed by Western blot analysis of cyclin A (Fig. 5B) and cyclin B1 (Fig. 5C) expression. This system also displayed a high level of PCTAIRE-1 activity in the S phase and G2 phase (Fig. 5D), thus confirming the observations made with Hs68 fibroblasts.

PCTAIRE-1 Phosphorylation. Signals such as phosphorylation are responsible for timing and coordinating cell cycle progression. In particular, cdks are phosphorylated at conserved residues that positively and negatively affect kinase activity (4). To determine whether changes in the phosphorylation state of PCTAIRE-1 correlate with fluctuations in kinase activity during cell cycle progression, we examined the phosphotyrosine content of the protein. For this examination, Hs68 cell extracts made during transition through G1 or upon release from the hydroxyurea block were immunoprecipitated with a monoclonal antibody to phosphotyrosine. PCTAIRE-1 was detected with a specific antibody upon resolution of the proteins by SDS-PAGE and transfer to PVDF membranes. The kinase appeared to be tyrosine phosphorylated in G1, whereas progression through the cell cycle upon release from the hydroxyurea block was marked by tyrosine dephosphorylation of the protein (Fig. 4D). High levels of tyrosine phosphorylation were also detected in G0 (data not shown). The fact that tyrosine phosphorylation correlates with PCTAIRE-1 inactivation and that a low level of activity is observed in G0 indicates that the kinase may play a significant role in proliferation rather than in differentiation. Localization of PCTAIRE-1. In addition to regulating the process of kinase activation, cyclins also confer substrate specificity to cdks (29) and contribute to determine their intracellular distribution (30). To assess the subcellular localization of PCTAIRE-1, we examined the protein by indirect immunofluorescence after ectopic expression in Swiss 3T3 cells. The kinase was clearly localized in the cytoplasm. Identical results were obtained upon staining cells with either anti-PCTAIRE-1 (Fig. 6B) or anti-HA antibody (Fig. 6C). The same subcellular distribution was observed in transfected COS-7 and Hs68 cells (data not shown). Endogenous PCTAIRE-1 protein could not be detected by indirect immunofluorescence in any of the cell lines tested including A549, A431, 293 human embryo kidney, HeLa, COS-7, Hs68, and NIH3T3 (data not shown). To examine the subcellular distribution of PCTAIRE-1 in more detail, we have used confocal microscopy. The kinase was found to be homogeneously distributed throughout the cell (Fig. 7). In some instances, PCTAIRE-1 staining was apparently more intense in the perinuclear region (Fig. 6). Time course expression experiments demonstrated that this was not a function of protein accumulation in the cell (data not shown). The subcellular distribution of PCTAIRE-1 prompted us to examine its possible colocalization with the cytoskeletal network. Costaining of PCTAIRE-1 with actincontaining structures by means of Alexa 594 phalloidin or with the tubulin network (Fig. 7) did not display any specific colocalization. Furthermore, colocalization of PCTAIRE-1 with other subcellular structures such as the endoplasmic reticulum, which was stained with dihexyloxyacarbocyanine iodide (DioC6), was negative (data not shown). The kinase subcellular distribution during progression through the cell cycle was examined upon ectopic expression of HA-tagged PCTAIRE-1 in HeLa cells and synchronization of the cells by double thymidine block. The degree of cell synchronization was controlled by flow cytometric analysis of DNA content and by costaining with cyclin antibodies.

Cell Growth & Differentiation

Fig. 4. Cell cycle-dependent PCTAIRE-1 kinase activity and tyrosine phosphorylation in Hs68 fibroblasts. Hs68 cells were made quiescent by serum deprivation (48 h) and restimulated with 10% FCS for 20 h. A, cell cycle progression was monitored by flow cytometric analysis as described in “Materials and Methods.” B, extracts made from synchronized Hs68 fibroblasts (200 mg) at different times during the transition through the cell division cycle were immunoprecipitated and assayed for kinase activity as described in “Materials and Methods.” G0 (Lane 1), early G1 (2 h; Lane 2), late G1 (8 h; Lane 3), G1-S phase (Lane 4), S phase (Lane 5) and G2 (Lane 6) phase extracts were immunoprecipitated with anti-PCTAIRE-1 antibody. S-phase extracts treated with no antibody (Lane 7) or immunoprecipitated with anti-cyclin A antibody (Lane 8) were used as negative and positive controls, respectively. The results of an experiment representative of three independent determinations are shown. C, an aliquot (15 mg) of the extracts used in A was probed with PCTAIRE-1 antibody to examine the level of endogenous PCTAIRE-1 expression during cell cycle progression. D, extracts (200 mg) made in G1 or upon release from the hydroxyurea block (see “Materials and Methods”) were immunoprecipitated using a monoclonal antiphosphotyrosine antibody. Proteins were resolved by SDS-PAGE and transferred to PVDF membrane. The membrane was probed with an anti-PCTAIRE-1 antibody and revealed with the ECL detection kit. G1 (Lane 1) or hydroxyurea block-released (Lane 2) Hs68 cells. G1 extract treated with no antibody was used as a negative control (Lane 3). The band below PCTAIRE-1 is nonspecifically detected by the antibody used to probe the membrane because it is also present in the negative control (Lane 3).

The representative expression and subcellular localization of cyclins (31) for each phase of the cell cycle are shown in the right panels of Fig. 8A. Whereas cyclin E and A are constitutively nuclear at the G1-S-phase transition and in S phase, respectively, cyclin F is distributed in both the nuclear and cytoplasmic compartment in G2. Cyclin B1, on the other hand, is localized in the cytoplasm during G2 and translocates to the nucleus at the G2-M-phase transition. The results presented in Fig. 8A clearly show that PCTAIRE-1 was excluded from the nucleus at all times examined. BrdUrd staining of the nuclei was used as an additional control for S phase, the time at which PCTAIRE-1 activity is maximal (Fig. 8B). Analysis of PCTAIRE-1 distribution during the cell cycle of Hs68 fibroblasts synchronized by serum starvation gave identical results (data not shown).

Discussion Progression through the cell division cycle is orchestrated by the timely activation of members of the cdk family. The cdc2 gene, whose human homologue was identified by complementation studies in the yeast S. pombe (3), encodes the prototype member of the family. Components of the cdk family have been isolated by PCR using degenerate oligo-

nucleotides (6). PCTAIRE-1, like the related molecules PCTAIRE-2 and PCTAIRE-3, is one such kinase displaying a high degree of homology to p34cdc2. PCTAIRE kinases are almost twice the size of other members of the family because of NH2- and COOH-terminal extensions. Some members of the cdk family are predominantly expressed in terminally differentiated cells (32), including PCTAIRE-2 (33). To investigate the role played by PCTAIRE-1 in the cell division cycle, we began by analyzing the kinase’s pattern of protein expression. The data obtained indicated that contrary to PCTAIRE-2 (33), PCTAIRE-1 is expressed across the entire panel of cell lines that we have examined. The fact that transformed cells display high levels of PCTAIRE-1 protein suggests that the kinase is likely to be involved in proliferation. Contrary to cdk5, which is widely expressed but displays kinase activity only in neuronal cells (34), PCTAIRE-1 appears to be active in both normal and transformed cells (see below). PCTAIRE-1 migrated in SDS polyacrylamide gels with an apparent Mr of 55,000, in agreement with the predicted size of the polypeptide (Mr 55,715). In some of the cell lines examined, we detected a slightly larger peptide, which has also been observed by others (23). The human PCTAIRE-1

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Fig. 5. Cell cycle-dependent PCTAIRE-1 kinase activity in HeLa cells. HeLa cells were synchronized by a double thymidine block. Cell cycle progression was monitored by flow cytometric analysis (A) and by the pattern of cyclin A (B) and cyclin B1 (C) expression. D, protein kinase activity was determined upon immunoprecipitation with anti-PCTAIRE-1 antibody as described in “Materials and Methods.” G1 (12 h after release), Lane 1; G1-S phase (0 h), Lane 2; S phase (4 h after release), Lane 3; G2 (8 h after release), Lane 4. S-phase extracts immunoprecipitated without an antibody (Lane 5) or with an anti-Cdk2 antibody (Lane 6) were used as negative and positive controls, respectively.

DNA sequence (GenBank accession number X66363) displays an ATG at 1124. This is very likely the site of initiation because it contains G in the critical 23 and 14 position and displays other features that are supposed to facilitate frame monitoring by ribosomes (35). Another ATG is located at position 2104 relative to the site, conforming to Kozak’s rule. However, this second ATG is followed by two stop codons at position 287 and 266, thus ruling out the possibility of initiation at this site. Accordingly, ectopic expression of HAtagged PCTAIRE-1 in COS-7 cells yielded a single protein of Mr 55,000, indicating that the ATG at position 1124 is the only site of initiation in this case. Western blot analysis carried out in the presence of the peptide used to immunize rabbits showed that a 10-fold excess of peptide could compete out both the large band and the small band, indicating that the two bands are immunologically related proteins. These two polypeptides may originate from differentially spliced PCTAIRE-1 mRNA (23). Differential splicing is a common event responsible for generating multiple isoforms of a protein that presumably play different roles in the cell. Such a phenomenon has been widely described for protein kinases (36 –38). The question of whether distinct PCTAIRE-1 isoforms play different roles in the cell will require additional studies. PCTAIRE-2 is a member of the PCTAIRE subgroup whose kinase activity was the first to be examined (33). This kinase

appears to efficiently phosphorylate histone H1. A recent report indicates that rat PCTAIRE-1 also displays such an activity (27). In both cases, the enzymes were derived from rat brain. We observed that immunopurified human PCTAIRE-1 displays activity on MBP and is capable of autophosphorylation. By introducing a single point mutation at residues involved in the process of catalysis, we were able to generate two distinct kinase-dead mutants and abolish MBP phosphorylation. This demonstrates that the kinase activity observed in our assay was intrinsic to PCTAIRE-1 and was not derived from a contaminating, associated kinase. As reported previously for one such mutant (39), ectopic expression of kinase-dead PCTAIRE-1 did not affect the pattern of cell cycle progression (data not shown). A highly purified GST fusion form of PCTAIRE-1 expressed in Sf-9 insect cells was devoid of kinase activity. The Sf-9/baculovirus system provides a valuable approach to elucidate signaling pathways because of its high homology with mammalian systems. In many instances, exogenously expressed proteins benefit from the high degree of signaling pathway conservation and are produced in an active form. This is the case for p70/ p85S6k (40), Akt/PKB (41), and other kinases requiring posttranslational modifications for activation. cdks, on the other hand, require both phosphorylation and interaction with cyclins to display activity. Studies conducted in Sf-9 cells expressing cdk4 showed that kinase activity was detected only upon coinfection with the regulatory subunit of the complex, indicating that cyclins were the limiting factor in the system (42). PCTAIRE-1 activation seems to conform to this mechanism because preincubation of the inactive form of recombinant GST-PCTAIRE-1 with cell extract obtained from exponentially growing human fibroblasts led to full activation of the kinase. This may result from either posttranslational modification of PCTAIRE-1, productive interaction with a regulatory partner, or both. Among these possibilities, complex formation with an activating factor seems to be a necessary event, as shown by the lack of MBP and autophosphorylation activity upon the resolution of immunopurified PCTAIRE-1 in SDS gel followed by an in situ kinase assay. This indicates that PCTAIRE-1 kinase activity apparently depends on a noncovalent interaction with a regulatory component. These data also indicate that the presence of NH2and COOH-terminal extensions in PCTAIRE-1 does not compensate for the absence of a regulatory partner. Using HeLa cells metabolically labeled with [35S]methionine/[35S]cysteine, we were unable to detect proteins coprecipitating in stoichiometric amounts with PCTAIRE-1. Moreover, immunoprecipitation of either endogenous or ectopically expressed PCTAIRE-1, followed by Western blot analysis with antibodies to a panel of characterized and orphan cyclins, revealed that none of those proteins complex with the kinase in vivo. Similar results were obtained in pull-down studies when fibroblast cell extract was probed with highly purified GST-PCTAIRE-1. Evidence obtained in a two-hybrid screen indicated that mouse PCTAIRE-1 is capable of interacting with the 14-3-3h, u, and z proteins in vitro (26). These are proteins that bind to phosphorylated serine residues situated in a defined motif (43). Protein interaction detected in twohybrid screens often results from the presence of adapter

Cell Growth & Differentiation

Fig. 6. Subcellular localization of ectopically expressed PCTAIRE-1. Swiss 3T3 cells expressing HA-tagged PCTAIRE-1 were fixed and probed with PCTAIRE-1 (B) or HA antibody (C). A, Hoechst staining of DNA. Bar, 10 mm.

proteins in the host that function by bridging the bait to the prey. This was shown to be the case for p57Kip2/cyclin D1 (44). Interaction between PCTAIRE-1 and the 14-3-3 proteins was subsequently confirmed in vivo, although a demonstration that dephosphorylated PCTAIRE-1 is incapable of binding 14-3-3 proteins was lacking in this study (27). Moreover, a nonhomogeneous preparation of PCTAIRE-1 was used, suggesting that the results of PCTAIRE-1/14-3-3 protein interaction should be interpreted with caution. Although a consensus sequence for binding to 14-3-3 proteins is present at the COOH terminus of the kinase, we were not able to coimmunoprecipitate ectopically expressed, HA-tagged PCTAIRE-1 and 14-3-3 proteins in HeLa cells (data not shown). The question of whether such an interaction is specific to the neuronal system remains to be addressed. The activity of cdk family members is controlled by positive and negative phosphorylation at conserved residues (4). These events allow for a narrow window of cdk activation during cell cycle progression. PCTAIRE-1 displays a conserved Thr/Tyr motif in subdomain I of the kinase catalytic domain and a Ser in subdomain VIII at a position corresponding to Thr161 in p34cdc2. The Thr/Tyr motif is located in the ATP-binding pocket of cdks. Phosphorylation of these residues impairs binding of the nucleotide and de facto results in lack of phosphotransferase activity (4). Analysis of PCTAIRE-1 in synchronized Hs68 fibroblasts or HeLa cells showed that when cells emerge from quiescence and move through G1, the level of kinase activity is low. This reaches a minimum at the time of the G1 to S-phase transition. Conversely, PCTAIRE-1 displays maximal activation during S phase and G2. We found that the extent of PCTAIRE-1 activation inversely correlates with the content in phosphotyrosine, which is high in G0 and during progression through G1. As observed for other cdk family members, the PCTAIRE-1 level did not change during transition through the cell division cycle. The presence of stretches of basic residues at the NH2 terminus of human and murine PCTAIRE-1 has led to the suggestion that these may serve as nuclear localization signals (23). The low levels of PCTAIRE-1 protein in the cells prevented the detection of endogenous kinase in immunofluorescence studies. However, ectopically expressed HAtagged PCTAIRE-1 was readily detected with either anti-HA or anti-PCTAIRE-1 antibody. Indirect immunofluorescence revealed that the kinase is localized in the cytoplasm, and no change in the subcellular localization of PCTAIRE-1 was

observed during cell cycle progression. These data argue against a role for the presumed NH2-terminal nuclear localization signal in vivo. The staining pattern shown by PCTAIRE-1 prompted us to address its potential colocalization with structures of the cytoskeletal network. Analysis of PCTAIRE-1 distribution by confocal microscopy revealed that the regions of PCTAIRE-1 staining did not correspond to particular structures of the actin and tubulin network or the endoplasmic reticulum. The fact that PCTAIRE-1 activity is maximal when cells transit through S phase, a time when the genome is replicated, and the kinase is constantly excluded from the nucleus indicates that PCTAIRE-1 is not actively involved in DNA replication. Additional studies will address the issue of whether PCTAIRE-1 supports events related to a correct transition through S phase.

Materials and Methods DNA Constructs. The plasmid pBSSK-PCTAIRE-1 was kindly provided by Greg H. Enders (Massachusetts General Hospital Cancer Center, Charlestown, MA). Sequencing of the insert revealed a single base mutation (G/A at position 727) with respect to the published sequence (GenBank accession number X66363). The PCTAIRE-1 open reading frame was subcloned into a pcDNA3 vector (Invitrogen) containing a HA epitope for transient expression in mammalian cells and into a modified pFbac1 vector (Life Technologies, Inc.) containing a GST tag for expression in Sf-9 insect cells. PCTAIRE-1 point mutants (D304N and K194A) were generated with the QuickChange site-directed mutagenesis kit (Stratagene) and controlled by DNA sequencing. Cell Culture, Transfection, and Synchronization. Human foreskin Hs68 fibroblasts (ATCC CRL 1635), COS-7 cells (ATCC CRL 1651), HeLa cells (ATCC CCL2), and Swiss 3T3 cells (ATCC CCL92) as well as wildtype (ATCC CCL75) and SV40-transformed (ATCC CCL75.1) WI38 fibroblasts were cultured at 37°C in DMEM supplemented with 10% FCS (Life Technologies, Inc.), 1 mM glutamine (Life Technologies, Inc.), and penicillin/streptomycin (100 units/ml and 100 mg/ml, respectively) on plastic dishes or acid-washed coverslips. Cells were seeded 16 –24 h before transfection and were transfected using the FuGENE 6 kit (2 mg plasmid/ 100-mm dish), as described by the supplier (Boehringer Mannheim). Hs68 cells were made quiescent by a 48-h serum deprivation and restimulated with 10% FCS for 20 h. Synchronization of Hs68 cells at the G1-S-phase boundary was obtained as described previously (28). Cell cycle progression was monitored by fluorescence-activated cell-sorting analysis as described below. HeLa cells were synchronized by a double thymidine block. Briefly, 4 3 105 cells were seeded in 100-mm plates and allowed to grow for 36 h. Thymidine (2 mM) was added to cultures for 14 h, and cells were released from the block by washing three times with medium. After 8 h, thymidine was added again. After a second treatment for 14 h, cells were released from the block and harvested at 4 h (S phase), 8 h (G2 phase), and 12 h (G1 phase). When immunofluorescence studies were performed, HeLa cells were transfected with HA-tagged PCTAIRE-1 24 h after seeding and subjected to a double thymidine block as described above. Jurkat cells were maintained in RPMI 1640 supplemented with

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Fig. 7. Comparison of PCTAIRE-1 distribution with the actin and tubulin network. Swiss 3T3 cells were fixed in formalin and permeabilized with Triton X-100 (A and B) or fixed with cold methanol (C and D) before probing with PCTAIRE-1 antibody (A and C), Alexa 594 phalloidin (B), or monoclonal tubulin antibody (D). Cells were observed under a confocal laser scanning microscope. Bar, 20 mm.

10% FCS. Before stimulation for 10 min with 2.5 mM H2O2, cells were grown overnight in RPMI 1640 containing 1% FCS. BrdUrd Labeling of Nuclei. The number of S-phase cells was determined using BrdUrd labeling (Boehringer Mannheim Kit 1296736) according to the manufacturer’s instructions. HeLa cells were released from the double thymidine block for 3 h and treated with the labeling reagent for 1 h. Anti-BrdUrd solution II was added together with anti-PCTAIRE-1 antiserum (1:300). Antimouse Alexa 568 (1:50; Molecular Probes, Eugene, OR) and antirabbit FITC (Amersham; 1:50) were used as secondary antibodies for the detection of BrdUrd and PCTAIRE-1, respectively. Ectopic Expression of PCTAIRE-1 in Sf-9 Cells and Pull-Down GST Analysis. GST-PCTAIRE-1 was produced following the instructions given in the Bac to Bac manual (Life Technologies, Inc.). Briefly, Sf-9 cells were infected with the virus for 96 h and centrifuged at low speed, and the resulting pellet was lysed in buffer A [50 mM Tris-HCl (pH 7.5), 1 mM DTT, 10 mM NaCl, 5 mM EDTA, 0.5 mM sodium orthovanadate, 0.1% Triton X-100, 1% glycerol, and 1 tablet/50-ml mixture of protease inhibitors from Life Technologies, Inc.]. The cell lysate was treated with a Dounce homogenizer at 4°C. The resulting homogenate was centrifuged at 4°C for 30 min at 15,000 3 g. GST-PCTAIRE-1 was purified using a glutathioneSepharose 4B column (Pharmacia), and the fusion protein was eluted with 10 mM reduced glutathione. Proteins were examined by Coomassie Blue staining. Western blotting was performed by using either affinity-purified polyclonal anti-PCTAIRE-1 (Upstate Biotechnology Inc.; 1 mg/ml) or antiGST antibody (Santa Cruz Biotechnology). Before analyzing kinase activity, GST-PCTAIRE-1 was dialyzed against buffer B [25 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and 1 tablet/50-ml protease inhibitor mixture]. For pull-down studies, 5 mg of GST-PCTAIRE-1 were incubated in the presence or absence of 50 mg of fibroblast cell extract for 1 h at 4°C in 100 ml of buffer C [50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM sodium PPi, 15 mM p-nitrophenyl phosphate, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium orthovanadate, and 1% NP40]. Interacting proteins were precipitated using 10 ml of glutathione-Sepharose 4B (Pharmacia) at 4°C and washed three times with 1 ml of ice-cold buffer C. Proteins were resolved on a 10% Laemmli gel and transferred to a PVDF membrane (Millipore).

Fig. 8. Subcellular distribution of HA-tagged PCTAIRE-1 during cell cycle progression. HeLa cells were transfected with HA-tagged PCTAIRE-1 and synchronized by a double thymidine block. A, cells were fixed in cold methanol before probing with HA or PCTAIRE-1 antibody (left panels) and cyclin antibody (right panels). B, cells in S phase were labeled with BrdUrd (red) and stained with PCTAIRE-1 antibody (green). Bar, 10 mm.

Western Blotting, Immunoprecipitations, and Protein Kinase Assay. Cell extracts were prepared in ice-cold buffer C. Extracts were centrifuged at 14,000 rpm for 10 min at 4°C in an Eppendorf centrifuge. Protein concentration was determined with a BCA protein assay kit (Pierce). For Western blotting, equal amounts of protein (15 mg) were resolved on 10% Laemmli gel and transferred to PVDF. The resulting membrane was probed with PCTAIRE-1 polyclonal antibody and revealed using the ECL system (Amersham). The specificity of the antibody was

Cell Growth & Differentiation

examined in competition experiments in which a 10-fold excess (w/w) of antigenic peptide (residues 484 – 496; Eurogentec, Seraing, Belgium) was used. To detect cyclins, an anti-cyclin B1 monoclonal antibody (PharMingen) and a rabbit polyclonal anti-cyclin A antiserum (a kind gift of T. Lorca; Centre de Recherche de Biochimie Macromoleculaire, Montpellier, France) were used. Immunoprecipitations were carried out for 3 h at 4°C in buffer C using equal amounts of total protein (100 –300 mg), 2 mg of anti-PCTAIRE-1, 30 ml of anti-HA monoclonal antibody (12CA5; a gift from Y. Nagamine; Friedrich Miescher Institute, Basel, Switzerland), or 1 mg of anti-cyclin A antibody (C-19; Santa Cruz Biotechnology). Antibodies were immobilized on protein A- or protein G-Sepharose beads, and the resin washed three times with 1 ml of ice-cold buffer C. For kinase assays, extracts were processed as described above, except that the beads were also washed with 1 ml of buffer D (20 mM HEPES, 10 mM MgCl2, and 1 mM DTT). Beads were resuspended in a total volume of 25 ml of buffer D containing 5 mM ATP, 0.25 mCi of [g-32P]ATP (Amersham), and 0.5 mg/ml MBP (Sigma). MBP phosphorylation was analyzed by SDS-PAGE on a 15% Laemmli gel. Labeled bands were visualized by autoradiography or with a PhosphorImager (Molecular Dynamics). For in situ detection of kinase activity, 0.5 mg/ml MBP was added to the gel just before polymerization, which was performed as described previously (45), and kinase activity was measured as described previously (45). Tyrosine Phosphorylation Analysis. Extracts derived from synchronized fibroblasts (200 mg) were incubated with 5 mg of antiphosphotyrosine antibody (4G10) in buffer C for 3 h on ice before the addition of 20 ml of protein G-Sepharose for 1 h. Beads were washed three times with 1 ml of ice-cold buffer C and resuspended in 53 concentrated buffer E [80 mM Tris (pH 6.8), 10% glycerol, 2% SDS, 0.2% bromphenol blue, and 0.1 M DTT]. Proteins were resolved on an 8% Laemmli gel, transferred to PVDF membrane, and stained with 0.1% Ponceau S solution. Western blot with anti-PCTAIRE-1 antibody was performed as described above. Flow Cytometric Analysis of DNA Content. Cells were harvested by trypsinization, fixed in 70% ethanol, and stored in fixative at 220°C until prepared for cell cycle analysis. Labeling of cells for flow cytometric analysis of cell cycle phases was performed according to established methods (46, 47). Briefly, upon fixation, samples were pelleted by centrifugation at 300 3 g for 15 min at 4°C. The supernatant was removed, and pellets were washed three times with PBS containing 0.1% sodium azide to remove the ethanol and rehydrate the cells. The samples were resuspended in 200 ml of PBS containing 1 mM EDTA and 100 mg/ml RNase A (Sigma R5000 preboiled for 5 min) and incubated for 30 min at 37°C. The volume was adjusted to 1 ml with PBS containing final concentrations of 100 mg/ml RNase A, 100 mg/ml propidium iodide (Molecular Probes), and 0.1% Triton X-100. Final cell concentrations were 0.5–1 3 106 cells/ml. Samples were placed in the dark overnight at 4°C. Just before analysis, cell aggregates were broken up using a 25-gauge needle and a 1-ml syringe by gentle (three to four times) trituration. Acquisition and analysis were performed using a flow cytofluorometer (FACSCalibur and Cell Quest software; Becton Dickinson, Mountain View, CA). Data were acquired using linear amplification of FL-2 (excitation, 488 nm; emission, BP 585 nm/42) area measurement, pulse processing (area versus width) to gate on single events, and low flow rate, with the total event rate not exceeding 300 events/s. Data acquisition was set to stop after 9 min or after a minimum of 10,000 events had been collected in the single events region. List mode files contained all events detected above a threshold of DNA content $ 10% of the G0/G1 content. The G0/G1 peak for an untreated cycling control was set at channel 235, which yielded at least 200 channels for S-phase resolution. Immunofluorescence and Confocal Microscopy. Indirect immunofluorescence experiments were performed with cells expressing HAtagged PCTAIRE-1 that were grown on acid-washed glass coverslips. Briefly, cells were washed with PBS, fixed in 220°C methanol for 4 min or in 3.7% formalin in PBS for 5 min, and subsequently permeabilized with 0.2% Triton X-100 in PBS for 4 min. Coverslips were incubated for 60 min at 37°C in 1% BSA-PBS containing the primary antibody mix (a 1:50 dilution of each antibody) indicated in the figure legends. Purified rabbit polyclonal antisera to cyclin E (SC-198), cyclin A (SC-596), and cyclin F (SC-952) were obtained from Santa Cruz Biotechnology, whereas the monoclonal antibody to cyclin B1 was obtained from PharMingen. After washing with PBS, fluorescein-conjugated donkey antirabbit IgG (Amersham; 1:50 dilution in 1% BSA-PBS) combined with biotinylated sheep

antimouse IgG (Amersham; 1:200 dilution in 1% BSA-PBS) was added to the cells for an additional 45 min at 37°C. Cells were washed with PBS and finally incubated with a mixture of streptavidin-Texas Red (Amersham; 1:200 dilution in 1% BSA-PBS) and Hoechst dye (Sigma; 2 mg/ml) for 30 min. Coverslips were mounted in FluorSave (Calbiochem). Cells were observed with an Axioplan microscope (Zeiss) equipped with a 100-W HBO lamp for fluorescence. High-resolution pictures were taken with oil immersion lenses (63XNA). Images were captured with a color-chilled, three charge-coupled device camera (Hamamatsu, model C5810). For dual labeling of PCTAIRE-1 and microtubule or actin networks, we used either a monoclonal antitubulin antibody (Sigma, 1:200 dilution in 1% BSA-PBS) or Alexa 594 phalloidin (Molecular Probes; 1:20 dilution in 1% BSA-PBS) mixed with anti-PCTAIRE-1 antibody. An inverted Leica microscope equipped with the TCS confocal system and an Ar/Kr laser was used to obtain images from immunostained cells through a 1003 objective with numerical aperture 1.4. Stacks of images were analyzed using the Imaris program (BitpLane AG, Zurich, Switzerland) implemented on a Silicon Graphics computer.

Acknowledgments We thank Drs. J. Martin-Perez and B. Schott for helpful suggestions and Drs. T. Lorca (Centre de Recherche de Biochimie Macromoleculaire, Montpellier, France), Y. Nagamine (Friedrich Meischer Institute, Basel, Switzerland), and S. Ruetz (Novartis Pharma, Basel, Switzerland) for providing reagents and cells. We are also indebted to Dr. S. Kaech for assistance with immunofluorescence. Finally, we thank Drs. D. Fabbro (Novartis Pharma, Basel, Switzerland) and H. A. Lane (Friedrich Meischer Institute, Basel, Switzerland) for critical reading of the manuscript.

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