Functional characterization of human PFTK1 as a cyclin-dependent

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 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 (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 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. 3d), p21Cip1 is necessary for its association with PFTK1. It is well suggesting that at least part of the cellular PFTK1 is associated documented that the CDK-binding region is composed of amino with CCND3. These data convincingly demonstrate that the acids 42–82 on p21Cip1 (22). Our observation of PFTK1 binding association between PFTK1 and CCND3 is not only specific but to p21Cip1 through the CDK-binding domain of p21Cip1 provided also physiologically relevant. the first evidence that PFTK1 might be a CDK. On the other hand, another CDK inhibitor p16INK4a failed to interact with PFTK1 Is an Active Kinase, and Its Activity Is Enhanced by CCND3 and PFTK1 although it associated with its known binding partner Inhibited by p21Cip1. Previously, human PFTK1 has not been examined for its kinase activity and potential substrates. Stable cell lines expressing mouse PFTK1 were used by Besset et al. to examine its kinase activity toward different substrates. Although Leu-Trp- Leu-Trp-His-(3AT+) several substrates, including histone H1, casein, p56 Rb protein, neurofilaments, and myelin basic protein have been examined, none of them was found to be phosphorylated by mPFTK1 (3). The discovery of CCND3 and p21Cip1 as PFTK1-associated factors prompted us to further investigate the in vitro kinase activity of PFTK1. The 293T cells were transfected with PFTK1 or PFTK1 (K146A), in which the invariant lysine in subdomain II critical for ATP binding was mutated (25). Immunoprecipi- tated PFTK1 was subjected to an in vitro kinase assay, followed by SDS/PAGE and visualized by autoradiography. An Ϸ50-kDa 32P-labeled band corresponding to PFTK1 was observed only when the wild-type protein was immunoprecipitated (Fig. 4a). Taken together, these observations are consistent with PFTK1 being a bona fide protein kinase with intrinsic kinase activity. 1. pGB-PFTK1+pACT2 Furthermore, we investigated whether the kinase activity of 2. pGB+pACT2-p21 PFTK1 is regulated by CCND3 and p21Cip1. PFTK1 and CCND3 were cotransfected with p21Cip1 or its fragments in 293T cells. 3. pGB+pACT2-CCND3 Our data revealed that the autophosphorylation activity of 4. pGB-PFTK1+pACT2-p21 PFTK1 could be enhanced and inhibited by CCND3 and p21Cip1, Cip1 5. pGB-PFTK1+pACT2-CCND3 respectively (Fig. 4b). The inhibition by p21 was specific, because a truncated version of p21Cip1 (p21Cip1-D3), which failed 6. Positive Control to interact with PFTK1, did not show any inhibitory effect on

Cip1 PFTK1 activity. This result suggests that the association of Fig. 1. PFTK1 interacts with p21 and CCND3 in the yeast two-hybrid assay. Cip1 pGB or pGB-PFTK1 was cotransformed with pACT2, pACT2-p21Cip1, or pACT2- p21 with PFTK1 is required to exert the inhibitory effect of Cip1 CCND3 into the yeast strain Y190 along with a positive control pair, and p21 . Taken together, we conclude that PFTK1 is an active

tranformants were streaked on an SD/Leu-Trp plate (Left) and an SD/Leu-Trp- kinase whose activity is enhanced by CCND3 and inhibited by BIOCHEMISTRY His plate with 3-AT (Right). ␤-Galactosidase assay was also performed (Upper). p21Cip1.

Shu et al. PNAS ͉ May 29, 2007 ͉ vol. 104 ͉ no. 22 ͉ 9249 Downloaded by guest on September 28, 2021 a -N b -N c Flag-PFTK1 FL D1 D2 N Myc-p21 + + + + -PFTK1-PFTK1 -PFTK1-PFTK1 PFTK1 Flag Flag Flag Flag Kinase domain PFTK1-D1 PFTK1 PFTK1 PFTK1 PFTK1-D2 1 115 134 230 401 451 PFTK1-N PFTK1-N α PFTK1-N INPUT, IB:α-Flag INPUT, IB:α-Flag INPUT, IB: -Flag FL p21 endo p21 endo p21 α α α α α D1 INPUT, IB: -Myc IP: -p21, IB: -p21 IP: -Flag, IB: -p21 PFTK1 PFTK1 PFTK1 D2 PFTK1-D1 PFTK1-N IP:α-p21, IB:α-Flag IP:α-Flag, IB:α-Flag N IP:α-Myc, IB:α-Flag

d Cdk inhibitory domain Myc-p21 FL D1 D2 D3 C N Cyclin binding Cdk binding Flag-PFTK1 + + + + + + PFTK1 120406091164 p21 INPUT, IB: α-Flag FL p21 D1 fragments D2 INPUT, IB:α-Myc D3 PFTK1 C IP: α-Myc, IB: α-Flag N

e Flag-PFTK1 + + + - - f Flag-PFTK1 + + + + HA-CDK4 - - - + - HA-CCND3 - + + - Myc-p21 - + - - - Myc-p21 - - + + Myc-p16 - - + + + PFTK1 PFTK1 IB: α-Flag CDK4 INPUT CCND3 INPUT, IB:α-Flag , α-HA α p21 IB: -HA p16 p21 INPUT, IB: α-Myc IB: α-Myc PFTK1 PFTK1 CDK4 IB: α-Flag HRP IP: α-Myc IB: α-Flag HRP α-HA HRP IP: α-Flag CCND3 p21 IB: α-HA HRP p16 p21 IP: α-Myc, IB: α-Myc HRP IB: α-Myc HRP

Fig. 2. PFTK1 forms a ternary complex with p21Cip1 and CCND3. (a) The 293T cells were transfected with indicated constructs of PFTK1. Lysates were immunoprecipitated with anti-p21Cip1 and blotted with anti-p21Cip1 and anti-Flag. (b) Anti-Flag was used to immunoprecipitate the same lysates used in a. The immunoprecipitants were blotted with anti-p21Cip1 and anti-Flag. (c Left) Schematic representation of Flag-tagged PFTK1 truncations. (c Right) The 293T cells were transfected with indicated constructs. Lysates were immunoprecipitated with anti-Myc and blotted with anti-Flag. Lysates were also blotted with anti-Myc and anti-Flag. (d Left) Schematic representation of Myc-tagged p21Cip1 truncations. (d Right) PFTK1 interacts with full-length p21Cip1, D1, D2, and N fragments of p21Cip1.(e) The 293T cells were transfected with indicated constructs. Lysates were immunoprecipitated with anti-Myc and blotted with anti-Myc, anti-Flag, and anti-HA. (f) The 293T cells were transfected with indicated constructs. Lysates were immunoprecipitated with anti-Flag. Both lysates and immunoprecipitants were blotted with anti-Flag-HRP, anti-HA-HRP, and anti-Myc-HRP separately.

PFTK1 Phosphorylates the Tumor Suppressor Rb. Rb has been shown established a cell line stably overexpressing PFTK1 by retroviral to be a substrate for all cyclin D-associated CDKs. We went on infection of U2OS cells. A control cell line using empty vector to test the possibility of Rb being a substrate of PFTK1–CCND3 was also established. We next evaluated their growth character- complex in vitro. In agreement with our previous finding, the istics by delineating the growth curve of these two stable cell lines phosphorylation of RbC137 (amino acids 792–928 of Rb) by synchronized at G1 with 0.5 mM L-mimosine. After incubation PFTK1 was marginal and greatly enhanced in the presence of with L-mimosine for 24 h, cells were released from drug treat- CCND3 (Fig. 4c), again supporting the notion that PFTK1 is a ment and harvested every 4 h for cell cycle analysis. As shown cyclin-dependent kinase. Moreover, the CCND3-dependent in Fig. 5a, cells with stably expressed PFTK1 entered S-phase PFTK1 phosphorylation of RbC137 was significantly inhibited by faster than control cells by FACS analysis after removal of L-mimosine, indicating that PFTK1 promotes cell cycle progres- p21Cip1 but not by its truncated fragment p21Cip1-D3 (Fig. 4c). sion. Overall, cells with PFTK1 stably expressed displayed a These data demonstrate that PFTK1 can phosphorylate Rb, at reduced doubling time compared with the control cell line. least in vitro, and this phosphorylation is enhanced by CCND3 However, there is no growth difference between the two cell and inhibited by p21Cip1. Moreover, endogenous PFTK1 protein lines released from G2/M arrest induced by 50 ng/ml nocodazole immunoprecipitated with an anti-PFTK1 antibody was also (data not shown), suggesting that PFTK1 may specifically pro- capable of phosphorylating Rb (Fig. 4d), further suggesting that mote G1 to S transition. This result is consistent with the Rb is a bona fide substrate of PFTK1. observation that PFTK1 could phosphorylate Rb to exert its function. Expression of PFTK1 Promotes the S-Phase Entry. The facts that PFTK1 could be a CDK and that it phosphorylates Rb prompted siRNA Knockdown of PFTK1 Results in G1 Arrest. To further verify the us to investigate the role of PFTK1 in cell cycle regulation. We above observation, we designed and tested siRNA against

9250 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703327104 Shu et al. Downloaded by guest on September 28, 2021 a b -SH N SY5Y - - 0h 4h 8h 12h 16h 20h 24h 293T HeLa U2OS U251 SK JurkatSH PFTK1 PFTK1 IB: mouse α-PFTK1 IB: mouse α-PFTK1 β β-actin -actin α β IB: α-β-actin IB: - -actin c d

Input PFTK1 IP CDK6 IP PFTK1 Input Mock OutputCCND3 Output IB:α-PFTK1 PFTK1 CDK6 IB: α-PFTK1 α IB: -CDK6 CCND3 CCND1 IB: α-CCND3 IB:α-CCND1 β-actin CCND3 IB: α-β-actin IB:α-CCND3

Fig. 3. Endogenous interaction between PFTK1 and CCND3. (a) Expression of endogenous PFTK1 in different cell lines. Lysates of different cell lines were blotted with monoclonal anti-PFTK1 and anti-␤-actin. (b) U2OS cells were arrested in G1/G0 phase by treatment with 0.5 mM L-mimosine for 24 h. Cells were then released and harvested at indicated time points. Immunoblotting for endogenous PFTK1 was performed along with an anti-␤-actin blot. (c) Lysates from 1ϫ108 SH-SY5Y cells were immunoprecipitated with anti-PFTK1 or anti-CDK6, and the immunoprecipitants were blotted with anti-PFTK1, anti-CDK6, anti-CCND1, or anti-CCND3. (d) Lysates from SH-SY5Y cells were immunoprecipitated by using anti-CCND3 or mIgG as the control. The resulting supernatant was blotted with anti-PFTK1, anti-CCND3, or anti-␤-actin.

PFTK1, which showed efficient knockdown effect in SH-SY5Y the family members, p58 (PITSLRE), was reported to associate cells (Fig. 5b). We found that siRNA against PFTK1 induced a with a particular cyclin; coincidentally, that cyclin was CCND3 profound G1 arrest compared with the control siRNA in SH- (6). No other member of this family has been identified to have SY5Y cells (Fig. 5c). The percentage of G1 cells increased from a cyclin or a cyclin-like protein as a partner. Moreover, although 52.22% to 74.22%. Taken together, these data strongly suggest many associated factors of PFTK homologs have been identified that PFTK1 may represent a new member of CDK family in different species, none of them is cyclin-like, and their involved in cell cycle regulation. functional relevance remains to be demonstrated. Interestingly, the possibility of cyclin-based activation of Drosophila PFTK1, Discussion called L63, was raised by Stowers et al. (26). They showed that PFTK1 belongs to the CDK-like kinase family, which include L63 (G243A) failed to rescue the lethal phenotype in L63- mammalian PFTAIRE, PCTAIRE, PITSLRE, etc. Only one of deleted mutant animals when glycine 243, a residue conserved

a b

-PFTK1 -PFTK1* Flag-PFTK1 + + + + + + HA-CCND3 - + + + + + Flag Flag (K146A) p-PFTK1 Myc-p21 - - FL D2 D3 N Autoradiography p-PFTK1 PFTK1 IP:α-Flag, Autoradiography α IB: -Flag PFTK1 IP:α-Flag, IB:α-Flag c Flag-PFTK1 + + + + d 1 IP HA-CCND3 - + + + K Myc-p21 - - FL D3 IP C137 -PFT p-Rb IgG α IP:α-Flag, Autoradiography p-RbC137 PFTK1 Autoradiography IP:α-Flag, IB:α-Flag His-RbC137 PFTK1 IB:α-Rb IB: α-PFTK1

Fig. 4. PFTK1 is an active kinase that phosphorylates Rb. (a) The 293T cells transfected with indicated constructs were lysed, and lysates were immunopre- cipitated with anti-Flag. Half of the precipitates were blotted with anti-Flag, and the other half were used for kinase assay. (b) The kinase activity of PFTK1 is enhanced by CCND3 and inhibited by p21Cip1. The 293T cells transfected with indicated constructs were harvested and analyzed as described above. The p21Cip1 derivatives used were the same as described in Fig. 2d Left.(c) Phosphorylation of His-RbC137 by PFTK1. The 293T cells transfected with indicated constructs were harvested and analyzed as described above. Two micrograms of His-RbC137 protein was added to each reaction as the substrate. (d) Activity of endogenous PFTK1

in U2OS cells. U2OS cells (1ϫ108) were harvested and immunoprecipitated with mouse anti-PFTK1 (mouse IgG was used as the control). Half of the precipitates BIOCHEMISTRY were used for blotting with rabbit anti-PFTK1 (Lower), and the other half were used for kinase assay containing 2 ␮gofRbC137 protein as the substrate (Upper).

Shu et al. PNAS ͉ May 29, 2007 ͉ vol. 104 ͉ no. 22 ͉ 9251 Downloaded by guest on September 28, 2021 a 180 80 120 40 0 Cell number 240 180 60 120 0 U2OS stable DNA content Control PFTK1

IB: α-Flag b c SH-SY5Y SH-SY5Y Mock RNAi PFTK1 RNAi

SH-SY5Y G0/G1 G0/G1 G0/G1:52.22% G0/G1:74.22% S: 21.26% S: 15.36% G2/M:26.66% G2/M:10.55% mock RNAi PFTK1 RNAi PFTK1 α S IB: mouse -PFTK1 G2/M S β-actin G2/M IB: α-β-actin

Fig. 5. PFTK1 regulates G1/S transition of cell cycle. (a) Stably expressed PFTK1 promotes cell cycle progression. A stable PFTK1-expressing cell line (U2OS) and a control cell line were synchronized by 0.5 mM L-mimosine for 24 h. The drug-containing medium was removed, and fresh medium was added (marked as 0 h) to allow cells to exit from G0/G1 arrest. Cells were collected for FACS analysis every 4 h. Lysates were blotted with anti-Flag to confirm the expression of PFTK1. (b) siRNA-mediated knockdown of PFTK1 in SH-SY5Y cells. Cells were harvested 48 h after transfection. The knockdown effect of siRNA against PFTK1 was examined by using an anti-PFTK1 monoclonal antibody. The blot was also probed with anti-␤-actin antibody. (c) siRNA against PFTK1 leads to cell cycle arrest at G1. Representative histograms for cell cycle distribution in SH-SY5Y cells transfected with control siRNA (Left) and PFTK1-specific siRNA (Right) are shown.

for cyclin binding, was mutated. Here, we report that CCND3 is absence of cyclin D-CDK complexes. However, it is important to a specific regulatory subunit for PFTK1, and further confirm the note that knockout embryos of these strains show reduced body interaction between PFTK1 and CCND3 both in vitro and in vivo. size, highlighting the significance of these cyclin D–CDK complexes D-type cyclins, in association with CDKs, promote cell division in controlling cell growth and/or cell proliferation (27). Moreover, by inactivating negative regulators of S-phase gene expression. The the most drastic consequence of the absence of cyclin D–CDK catalytic activities of cyclin D-dependent kinases have been well complexes arises in tissues with high proliferative demand such as documented only in relationship to their phosphorylation and the fetal hematopoietic compartment. For example, CCND3-null T inactivation of the transcriptional corepressor activity of the Rb cell progenitors are severely impaired in their ability to undergo family members. Our observation that PFTK1–CCND3 complex pre-TCR expansion (28). Likewise, PFTK1–CCND3 active com- could phosphorylate Rb coincided with this notion. Moreover, the plexes may also couple various kinds of homeostatic signals (growth fact that PFTK1–CCND3–p21 could form a stable ternary complex factors, nutrients, oxygen, etc.) to cell cycle progression. In fact, makes PFTK1 fit well in the cyclin D-associated CDK category. Hashida et al. (29) reported that PFTK1 mRNA levels in the To date, almost all of the members of Cdc2-like protein kinases, euthyroid TRHϪ/Ϫ cerebellum supplemented with thyroid hor- such as PFTAIRE, PCTAIRE, PITSLRE, PITAIRE, KKIALRE, mone were significantly decreased compared with those in the wild PISSLRE, etc. have restricted tissue expression and appear to be type. Recently, Tang et al. (30) reported PFTK1 as one of the four devoid of cell cycle function. However, human PFTK1 is widely negative regulators of insulin-responsive glucose transport. These expressed in different tissues in contrast to the restricted expression findings also suggest a regulatory role of PFTK1 in response to of mPFTK1 in testis and the nervous system, which prompted us to particular extracellular signals in specific tissues. explore its role in cell cycle regulation. Our results demonstrate that In this study, we demonstrate that endogenous CCND3, but not PFTK1 impels the transition from G1 to S phase, not only in CCND1, interacts with endogenous PFTK1 in SH-SY5Y cells (Fig. established stable cell lines but also in unmodified SH-SY5Y cells 3c). CCND3 is the least studied member of the D-type cyclins. It as shown by our RNAi assay (Fig. 5). However, from this study, we shows the broadest expression pattern of all three D-type cyclins, cannot rule out the possibility that PFTK1 may also function in cell and its abundance in quiescent tissues suggests that it has dual roles differentiation in specific tissues. in proliferation and differentiation (31). Because CCND3 is ex- The recent generation of targeted gene-disruption mouse models pressed ubiquitously, it is not surprising that PFTK1, another for individual cyclin Ds and CDK4/6, and different combination of broadly expressed protein, acts as its binding partner. In addition, them, has revealed that a majority of cell types can arise in the CCND3’s selective binding to another CDK-related kinase has

9252 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703327104 Shu et al. Downloaded by guest on September 28, 2021 been reported (6). PFTK1’s specific pairing with CCND3 may PharMingen (San Diego, CA); polyclonal rabbit antibodies against suggest a link between PFTK1 and cancer cell growth. Like all CCND1 and CDK6 were gifts from Yue Xiong (University of D-type cyclins, CCND3 is overexpressed in many human cancers. North Carolina, Chapel Hill, NC). Studies by Sicinska et al. found that mice lacking CCND3 showed greatly reduced sensitivity to malignancies triggered by specific Cell Culture and Stable Transfectants. Cell lines were obtained from oncogenic pathways in immature T lymphocytes. This requirement ATCC (American Type Culture Collection, Manassas, VA). The for CCND3 also operates in human malignancies, because knock- 293T and U2OS cells were maintained in DMEM (PAA, Pasching, down of CCND3 inhibited proliferation of T cell acute lympho- Austria) and RPMI medium 1640 (PAA) supplemented with 10% blastic leukemia (T-ALL) cells (28). It would be intriguing to FBS (Invitrogen, Carlsbad, CA) respectively. SH-SY5Y cells were investigate whether the association of PFTK1 with CCND3 is maintained in MEM/F-12 (50/50 mixture; JRH, Lenexa, KS) sup- related to specific oncogenic pathways involving CCND3. plemented with 10% FBS. Cell line stably expressing PFTK1 was Our identification and characterization of CCND3 and p21Cip1 as generated by retroviral infection of U2OS cells. Briefly, a retroviral specific regulatory proteins of PFTK1 added a previously uniden- vector containing human PFTK1 cDNA with an N-terminal Flag- tified member to the CDK family. We propose that cell cycle tag, pCI-PG8, and pCI-VSV were cotransfected into 293T cells. Viral supernatants were collected and used to infect U2OS cells. regulation by PFTK1–CCND3 complex would presumably be Drug-resistant cells were then selected, cloned, and screened for tightly linked to the growth and stimulation of the cell. Further PFTK1 expression. investigation of how PFTK1 responds to particular extracellular signals will help us understand the fine-tuning of cell growth and Transfections, Immunoprecipitations, and Immunoblotting. Transfec- proliferation in specific tissues. tion, immunoprecipitation and Western blotting were carried out as Materials and Methods described (32). Yeast Two-Hybrid Analysis. The full-length human PFTK1 (Gen- In Vitro Kinase Assay. Kinase assay was performed as described (32). ࿝ Bank, NM 012395) cDNA was cloned into modified pGBKT7 Recombinant C-terminal retinoblastoma protein (His-RbC137) was vector and was used as the bait to screen three pACT2-human expressed and purified from a BL21 (DE3) E. coli strain. cDNA libraries (HeLa, human T/B lymphocyte, and human fetal brain). Positive interactions were verified by ␤-galactosidase assay. siRNA Analysis. RNA interference was performed in SH-SY5Y cells. The siRNA sequence used to target PFTK1 was from position 376 Mammalian Expression Constructs. Human PFTK1 and its derivative to 395 relative to the first nucleotide of the start codon of human cDNAs were cloned into a mammalian expression vector pEF with PFTK1 (5Ј-GGAUCUUAUGCUACAGUAUtt-3Ј). The se- an N-terminal Flag tag. Human p16INK4a, p21Cip1 cDNA, and its quence of control siRNA was 5Ј-UUCUCCGAACGUGU- truncations were cloned into a pCDEF3 vector with an N-terminal CACGUtt-3Ј. siRNAs were chemically synthesized by Shanghai Myc tag. Human CCND3 and CDK4 were cloned into a pCDEF3 GenePharma (Shanghai, China). vector with an N-terminal hemagglutinin (HA) tag. For making the plasmid used for retroviral infection of U2OS cells, Flag-tagged Cell Cycle Analysis. U2OS and SH-SY5Y cells at a density of 2.5ϫ105 PFTK1 was constructed into a pMXN vector. pCDEF3, pEF, per ml were cultured in the presence of 0.5 mM L-mimosine for 24 h pMXN, pCI-VSV, and pCI-PG8 vectors were gifts from Arthur to induce G1 cell cycle arrest. For siRNA analysis, cells were Weiss (University of California, San Francisco, CA). harvested 48 h after transfection with siRNA. Cells were then labeled with propidium iodide (PI) and analyzed by using a Antibodies. Anti-HA, anti-Flag, and anti-Myc monoclonal antibod- FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). ies were generated in-house; anti-␤-actin antibody was obtained from Sigma (St. Louis, MO); monoclonal anti-PFTK1 antibody was We thank Arthur Weiss for pCDEF3 vector and Lewis L. Lanier (University of California, San Francisco, CA) for pMX vector. This work generated by using His-PFTK1 expressed in Escherichia coli BL21 was supported by National Natural Science Foundation of China (NSFC) (DE3); polyclonal anti-PFTK1 antibody was generated by using a Grant 30430630, National Outstanding Youth Fund of NSFC Grant PFTK1 peptide (LPHFKPERFTLYSSKNLRQC); anti-p21Cip1 30225022, Chinese National 863 program Grant 2002BA711A01-08, and and anti-CCND3 antibodies were obtained from BD Biosciences Key Program of Basic Research, Shanghai Grant STC 05JC14087.

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