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Functional Characterization of Human PFTK1 As a Cyclin-Dependent Kinase

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Functional Characterization of Human PFTK1 As a Cyclin-Dependent Kinase Functional characterization of human PFTK1 as a cyclin-dependent kinase Fang Shu*, Shun Lv*, Yan Qin*, Xinlu Ma*, Xin Wang†, Xiaozhong Peng†, Ying Luo*†‡§, Bing-e Xu*, Xiaoqing Sun*‡, and Jun Wu*†‡§¶ *Shanghai Genomics, Inc., ‡Chinese National Human Genome Center, Shanghai, Zhangjiang Hi-Tech Park, Shanghai 201203, China; †National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, National Human Genome Center, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China; and §GNI, Ltd., 4-2-12 Toranomon, Tokyo 1050001, Japan Communicated by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, April 18, 2007 (received for review September 15, 2006) Cyclin-dependent kinases (CDKs) are crucial regulators of the PFTK1 is a Cdc2-related protein kinase, also named as eukaryotic cell cycle whose activities are controlled by associated PFTAIRE1 (17). The gene is highly expressed in the brain, cyclins. PFTK1 shares limited homology to CDKs, but its ability to pancreas, kidney, heart, testis, and ovary and minimally ex- associate with any cyclins and its biological functions remain pressed in the spleen and thymus. Some interacting proteins of largely unknown. Here, we report the functional characterization PFTAIRE have been reported in different species. For example, of human PFTK1 as a CDK. PFTK1 specifically interacted with cyclin L63, the Drosophila PFTAIRE1, interacts with two novel pro- D3 (CCND3) and formed a ternary complex with the cell cycle teins, PIF-1 and PIF-2 (PFTAIRE interacting factor-1 and -2) inhibitor p21Cip1 in mammalian cells. We demonstrated that the (18); two specific sets of endogenous cytosolic proteins of kinase activity of PFTK1 depended on CCND3 and was negatively Ϸ58–60 and 200–205 kDa have been reported to associate with regulated by p21Cip1. Moreover, we identified the tumor suppres- mPFTAIRE1 (19); KIAA0202 and 14-3-3 proteins were iden- sor Rb as a potential downstream substrate for the PFTK1/CCND3 tified as hPFTAIRE1-interacting proteins (20, 21). However, complex. Importantly, knocking down PFTK1 expression by using none of the interacting partners is cyclin-like, and the functional siRNA caused cell cycle arrest at G1, whereas ectopic expression of significance of these PFTAIRE-associated factors has not been PFTK1 promoted cell proliferation. Taken together, our data fully addressed. strongly suggest that PFTK1 acts as a CDK that regulates cell cycle To uncover the biological function of PFTK1, we conducted progression and cell proliferation. a complete yeast two-hybrid screening, from which we identified two PFTK1-associating factors as CCND3 and p21Cip1.We cyclin D3 ͉ p21 ͉ retinoblastoma ͉ cell cycle showed that these three proteins formed a stable complex in vivo, and p21Cip1 inhibited the CCND3-dependent activation of yclin-dependent kinases (CDKs) are crucial regulators of the PFTK1. Further functional characterization of human PFTK1 Ceukaryotic cell cycle. Cdc2 was identified as the first CDK demonstrated that PFTK1 acts as a CDK likely involved in cell that was essential for G1/S and G2/M transitions in Schizosac- cycle regulation. charomyces pombe (1). Since then, homologs in all species from Results yeast to mammals have been found. So far, 11 mammalian CDK family members have been described, and that number is in- Interactions of CCND3 and p21Cip1 with PFTK1 Were Identified by Yeast creasing rapidly (2). CDKs are a family of Ser/Thr protein Two-Hybrid Screening. Although PFTK1 orthologs have been kinases that all share a highly conserved motif, known as the identified in many species, including human, mouse, and Dro- PSTAIRE motif, located in subdomain III of the kinase domain. sophila, functionally relevant interacting partners of PFTK1 have This motif is involved in the binding of a CDK to a cyclin and has yet to be found. To find previously unidentified PFTK1- been used to classify other newly identified CDK-related kinases, associated factors, we performed a yeast two-hybrid screen using such as PCTAIRE, PITSLRE, PFTAIRE, PITAIRE, KKI- human PFTK1 as the bait. Eighty-two positive clones were Cip1 ALRE, PISSLRE, MAK, and MRK, etc. (3–12). The function isolated. Interestingly, 42 of them encoded p21 protein of these CDK-related kinases has not been fully understood. fragments and 6 of them encoded CCND3 protein fragments Intriguingly, despite their sequence homologies with CDKs, (Table 1). No other cyclin or cell cycle regulator was identified almost all of the members of this group appear to be devoid of in the screening, suggesting the specificity of these interactions. cell cycle functions, largely because of their restricted tissue Interactions between full-length proteins were confirmed by expressions and failure of identifying a regulatory cyclin partner. direct yeast two-hybrid analysis (Fig. 1). The activity of most CDKs requires the formation of holoen- zymes. These holoenzymes contain both regulatory (cyclin) and PFTK1 Forms a Ternary Complex with p21Cip1 and CCND3. To examine catalytic (CDK) subunits, and, sometimes, additional proteins. the in vivo interaction and physiological relevance between Cip1 Distinct cyclin–CDK complexes drive cells through different PFTK1 and p21 identified in the yeast two-hybrid screening, phases of the cell cycle (1). Typically, the D-type cyclins (cyclins we first conducted semiendogenous coimmunoprecipitation ex- D1, D2, and D3) act as growth factor sensors, forming active periments. Cell lysates prepared from 293T cells transfected with Cip1 kinases with CDKs in response to extracellular signals. The PFTK1 were immunoprecipitated with anti-p21 . As shown in mitogen-dependent accumulation of cyclin D-CDK (CDK4 and CDK6 in particular) holoenzymes triggers the phosphorylation Author contributions: F.S. and S.L. contributed equally to this work; F.S., S.L., Y.L., B.-e.X., of Rb, a tumor suppressor, and cancels the growth-repressive X.S., and J.W. designed research; F.S., S.L., Y.Q., and X.M. performed research; F.S., S.L., Y.Q., functions of hypophosphorylated Rb (1, 13, 14). X.M., X.W., X.P., Y.L., B.-e.X., X.S., and J.W. analyzed data; and F.S., S.L., B.-e.X., X.S., and In addition to positive regulation by cyclins, CDKs are regu- J.W. wrote the paper. lated by several subunits named CKIs (CDK inhibitors) that The authors declare no conflict of interest. associate physically with cyclin–CDK complexes to inhibit their Abbreviations: CDK, cyclin-dependent kinase; CCND3, cyclin D3; CIP, CDK-interacting pro- activities and promote cell cycle arrest or delay. CKIs include tein; RB, retinoblastoma. INK4 and Cip/Kip family members (15). Among them, p21Cip1 ¶To whom correspondence should be addressed. E-mail: [email protected]. is mostly documented as a universal inhibitor of CDKs (16). © 2007 by The National Academy of Sciences of the USA 9248–9253 ͉ PNAS ͉ May 29, 2007 ͉ vol. 104 ͉ no. 22 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703327104 Downloaded by guest on September 28, 2021 Table 1. Yeast two-hybrid screening of PFTK1 protein CDK4 readily, showing the specificity of the PFTK1–p21Cip1 No. of positive clones/total interaction (Fig. 2e). Positive clone no. of positive clones GenBank accession no. The association between CCND3 and PFTK1 was relatively weak (Fig. 2f). However, the association between PFTK1 and p21cip1 42/82 NM࿝000389 CCND3 was markedly enhanced in the presence of p21Cip1. The Cyclin D3 6/82 NM࿝001760 result is reminiscent of the previous finding that p21Cip1 helps the PFTK1-interacting proteins were identified by using yeast two-hybrid formation of CDK–cyclin complexes during cell cycle regulation Cip1 screening. As indicated, 82 positive clones were isolated. Forty-two clones (23, 24). The p21 association with PFTK1 was independent encode p21cip1, and six encode cyclin D3. of the presence of CCND3 (Fig. 2f), again consistent with the observation that CKIs can bind to both monomeric CDKs and cyclin-bound CDKs and also use multiple mechanisms to inhibit Fig. 2a, PFTK1 interacted with endogenous p21Cip1. In a recip- CDKs and halt the progression of the cell cycle. rocal experiment, we also observed that endogenous p21Cip1 coimmunoprecipitated with ectopically expressed PFTK1 (Fig. Endogenous Interaction Between PFTK1 and CCND3. To examine the 2b). Taken together, our data demonstrate a specific interaction endogenous association between PFTK1 and CCND3, we gen- between PFTK1 and p21Cip1. erated a PFTK1 monoclonal antibody. As shown in Fig. 3a, To further map the p21Cip1-interacting region of PFTK1, we the PFTK1 antibody detected a specific protein band with the predicted molecular mass of PFTK1 in different cell lines. The constructed additional PFTK1 fragments (Fig. 2c Left), includ- expression level of PFTK1 stayed relatively constant during ing D1 (115–451 aa), D2 (230–451 aa), and N (1–134 aa), and the cell cycle (Fig. 3b), similar to the pattern seen with other transfected each cDNA construct into 293T cells together with Cip1 Cip1 CDKs. We then used the anti-PFTK1 antibody to immunopre- p21 . As shown in Fig. 2c, p21 was coimmunoprecipitated cipitate PFTK1 in the neuroblastoma cell line SH-SY5Y. We with full-length PFTK1 and its D1 derivative but not D2 and N, Cip1 could readily detect CCND3 in the PFTK1 immunoprecipitates, demonstrating that the kinase domain of PFTK1 was the p21 - indicating that these proteins indeed interact in vivo (Fig. 3c). In interacting region, in which amino acids 115 to 230 are necessary addition, this association appears specific, because we could not Cip1 for binding. Similarly, we constructed p21 truncation frag- detect any endogenous cyclin D1 (CCND1) in the PFTK1 ments (Fig. 2d Left), including D1 (20–164 aa), D2 (40–164 aa), precipitates. In contrast, CDK6 was coimmunoprecipitated with D3 (60–164 aa), C (91–164 aa), and N (1–91 aa). As shown in Fig. both CCND1 and CCND3 (Fig. 3c). Moreover, we used an 2d, PFTK1 interacted with D1, D2, and N fragments of p21Cip1, anti-CCND3 antibody to immunodeplete CCND3 from cells indicating that the region between amino acids 40 and 60 within lysates and found that the PFTK1 level was decreased (Fig.
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