A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability
2016; Elsevier BV; Volume: 291; Issue: 51 Linguagem: Inglês
10.1074/jbc.m116.753947
ISSN1083-351X
AutoresCorine St. Gelais, Sun Hee Kim, Lingmei Ding, Jacob S. Yount, Dmitri N. Ivanov, Paul Spearman, Li Wu,
Tópico(s)HIV Research and Treatment
ResumoSAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr592. Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells. SAMHD1 (sterile α motif and HD domain-containing protein 1) is a mammalian protein that regulates intracellular dNTP levels through its hydrolysis of dNTPs. SAMHD1 functions as an important retroviral restriction factor through a mechanism relying on its dNTPase activity. We and others have reported that human SAMHD1 interacts with the cell cycle regulatory proteins cyclin A, CDK1, and CDK2, which mediates phosphorylation of SAMHD1 at threonine 592, a post-translational modification that has been implicated in abrogating SAMHD1 restriction function and ability to form stable tetramers. Utilizing co-immunoprecipitation and co-localization approaches, we show that endogenous SAMHD1 is able to interact with the cyclin A-CDK1-CDK2 complexin monocytic THP-1 cells and primary monocyte-derived macrophages. Sequence analysis of SAMHD1 identifies a putative cyclin-binding motif found in many cyclin-CDK complex substrates. Using a mutagenesis-based approach, we demonstrate that the conserved residues in the putative cyclin-binding motif are important for protein expression, protein half-life, and optimal phosphorylation of SAMHD1 at Thr592. Furthermore, we observed that SAMHD1 mutants of the cyclin-binding motif mislocalized to a nuclear compartment and had reduced ability to interact with cyclin A-CDK complexes and to form the tetramer. These findings help define the mechanisms by which SAMHD1 is phosphorylated and suggest the contribution of cyclin binding to SAMHD1 expression and stability in dividing cells. SAMHD1 (sterile α motif and HD domain containing protein 1) is the only mammalian protein characterized as a dNTP triphosphohydrolase (dNTPase) 2The abbreviations used are: dNTPase, dNTP triphosphohydrolase; co-IP, coimmunoprecipitation; MDM, monocyte-derived macrophages; hpt, h post-transfection. 2The abbreviations used are: dNTPase, dNTP triphosphohydrolase; co-IP, coimmunoprecipitation; MDM, monocyte-derived macrophages; hpt, h post-transfection. enzyme (1.Goldstone D.C. Ennis-Adeniran V. Hedden J.J. Groom H.C. Rice G.I. Christodoulou E. Walker P.A. Kelly G. Haire L.F. Yap M.W. de Carvalho L.P. Stoye J.P. 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Kodigepalli K.M. Wu L. Regulation of deoxynucleotide metabolism in cancer: novel mechanisms and therapeutic implications.Mol. Cancer. 2015; 14: 176Crossref PubMed Scopus (74) Google Scholar). SAMHD1 has been characterized as a viral restriction factor that blocks the replication of several retroviruses, including HIV-1, and DNA viruses in quiescent cells such as resting T cells, macrophages, and dendritic cells (2.Hrecka K. Hao C. Gierszewska M. Swanson S.K. Kesik-Brodacka M. Srivastava S. Florens L. Washburn M.P. Skowronski J. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein.Nature. 2011; 474: 658-661Crossref PubMed Scopus (914) Google Scholar, 3.Laguette N. Sobhian B. Casartelli N. Ringeard M. Chable-Bessia C. Ségéral E. Yatim A. Emiliani S. Schwartz O. Benkirane M. 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Restriction of diverse retroviruses by SAMHD1.Retrovirology. 2013; 10: 26Crossref PubMed Scopus (114) Google Scholar). Some studies have reported that SAMHD1 binds nucleic acids (18.Beloglazova N. Flick R. Tchigvintsev A. Brown G. Popovic A. Nocek B. Yakunin A.F. Nuclease activity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restriction.J. Biol. Chem. 2013; 288: 8101-8110Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 19.Tüngler V. Staroske W. Kind B. Dobrick M. Kretschmer S. Schmidt F. Krug C. Lorenz M. Chara O. Schwille P. Lee-Kirsch M.A. Single-stranded nucleic acids promote SAMHD1 complex formation.J. Mol. Med. 2013; 91: 759-770Crossref PubMed Scopus (64) Google Scholar20.Goncalves A. Karayel E. Rice G.I. Bennett K.L. Crow Y.J. Superti-Furga G. Bürckstümmer T. SAMHD1 is a nucleic-acid binding protein that is mislocalized due to Aicardi-Goutieres syndrome-associated mutations.Hum. Mutat. 2012; 33: 1116-1122Crossref PubMed Scopus (107) Google Scholar). SAMHD1 has also been described to function as a ribonuclease (21.Ryoo J. Choi J. Oh C. Kim S. Seo M. Kim S.Y. Seo D. Kim J. White T.E. Brandariz-Nuñez A. Diaz-Griffero F. Yun C.H. Hollenbaugh J.A. Kim B. Baek D. et al.The ribonuclease activity of SAMHD1 is required for HIV-1 restriction.Nat. Med. 2014; 20: 936-941Crossref PubMed Scopus (218) Google Scholar), and it has been suggested that degradation of incoming viral genomic RNA is the primary mode of restriction against HIV-1 and other retroviruses (21.Ryoo J. Choi J. Oh C. Kim S. Seo M. Kim S.Y. Seo D. Kim J. White T.E. Brandariz-Nuñez A. Diaz-Griffero F. Yun C.H. Hollenbaugh J.A. Kim B. Baek D. et al.The ribonuclease activity of SAMHD1 is required for HIV-1 restriction.Nat. Med. 2014; 20: 936-941Crossref PubMed Scopus (218) Google Scholar, 22.Choi J. Ryoo J. Oh C. Hwang S. Ahn K. SAMHD1 specifically restricts retroviruses through its RNase activity.Retrovirology. 2015; 12: 46Crossref PubMed Scopus (60) Google Scholar). However, this finding remains controversial, and other studies have failed to identify RNase activity (23.Wittmann S. Behrendt R. Eissmann K. Volkmann B. Thomas D. Ebert T. Cribier A. Benkirane M. Hornung V. Bouzas N.F. Gramberg T. Phosphorylation of murine SAMHD1 regulates its antiretroviral activity.Retrovirology. 2015; 12: 103Crossref PubMed Scopus (39) Google Scholar, 24.Seamon K.J. Sun Z. Shlyakhtenko L.S. Lyubchenko Y.L. Stivers J.T. SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.Nucleic Acids Res. 2015; 43: 6486-6499Crossref PubMed Scopus (96) Google Scholar25.Antonucci J.M. St. Gelais C. de Silva S. Yount J.S. Tang C. Ji X. Shepard C. Xiong Y. Kim B. Wu L. SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity.Nat. 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Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1.J. Virol. 2013; 87: 11516-11524Crossref PubMed Scopus (123) Google Scholar29.St. Gelais C. de Silva S. Hach J.C. White T.E. Diaz-Griffero F. Yount J.S. Wu L. Identification of cellular proteins interacting with the retroviral restriction factor SAMHD1.J. Virol. 2014; 88: 5834-5844Crossref PubMed Scopus (74) Google Scholar). The HD domain of SAMHD1 contains the catalytic site of the dNTPase enzyme (1.Goldstone D.C. Ennis-Adeniran V. Hedden J.J. Groom H.C. Rice G.I. Christodoulou E. Walker P.A. Kelly G. Haire L.F. Yap M.W. de Carvalho L.P. Stoye J.P. Crow Y.J. Taylor I.A. Webb M. HIV-1 restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase.Nature. 2011; 480: 379-382Crossref PubMed Scopus (610) Google Scholar, 30.Ji X. Wu Y. Yan J. Mehrens J. Yang H. DeLucia M. Hao C. Gronenborn A.M. Skowronski J. Ahn J. Xiong Y. Mechanism of allosteric activation of SAMHD1 by dGTP.Nat. Struct. Mol. Biol. 2013; 20: 1304-1309Crossref PubMed Scopus (107) Google Scholar). Tetramer formation is required for dNTPase activity and HIV-1 restriction (31.Yan J. Kaur S. DeLucia M. Hao C. Mehrens J. Wang C. Golczak M. Palczewski K. Gronenborn A.M. Ahn J. Skowronski J. Tetramerization of SAMHD1 is required for biological activity and inhibition of HIV infection.J. Biol. Chem. 2013; 288: 10406-10417Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 32.Arnold L.H. Groom H.C. Kunzelmann S. Schwefel D. Caswell S.J. Ordonez P. Mann M.C. Rueschenbaum S. Goldstone D.C. Pennell S. Howell S.A. Stoye J.P. Webb M. Taylor I.A. Bishop K.N. Phospho-dependent regulation of SAMHD1 oligomerisation couples catalysis and restriction.PLoS Pathog. 2015; 11: e1005194Crossref PubMed Scopus (76) Google Scholar), although it has also been reported that oligomerization-deficient SAMHD1 mutants are capable of restricting HIV-1 (33.Brandariz-Nuñez A. Valle-Casuso J.C. White T.E. Nguyen L. Bhattacharya A. Wang Z. Demeler B. Amie S. Knowlton C. Kim B. Ivanov D.N. Diaz-Griffero F. Contribution of oligomerization to the anti-HIV-1 properties of SAMHD1.Retrovirology. 2013; 10: 131Crossref PubMed Scopus (29) Google Scholar). Phosphorylation of SAMHD1 at Thr592 is involved in negative regulation of SAMHD1-mediated HIV-1 restriction; however, there are conflicting data on whether dNTPase function is impaired by Thr592 phosphorylation (26.Cribier A. Descours B. Valadão A.L. Laguette N. Benkirane M. Phosphorylation of SAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1.Cell Rep. 2013; 3: 1036-1043Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 27.White T.E. Brandariz-Nuñez A. Valle-Casuso J.C. Amie S. Nguyen L.A. Kim B. Tuzova M. Diaz-Griffero F. The retroviral restriction ability of SAMHD1, but not its deoxynucleotide triphosphohydrolase activity, is regulated by phosphorylation.Cell Host Microbe. 2013; 13: 441-451Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar28.Welbourn S. Dutta S.M. Semmes O.J. Strebel K. Restriction of virus infection but not catalytic dNTPase activity is regulated by phosphorylation of SAMHD1.J. Virol. 2013; 87: 11516-11524Crossref PubMed Scopus (123) Google Scholar, 34.Tang C. Ji X. Wu L. Xiong Y. Impaired dNTPase activity of SAMHD1 by phosphomimetic mutation of Thr-592.J. Biol. Chem. 2015; 290: 26352-26359Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 35.Arnold L.H. 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Cyclin A is required at two points in the human cell cycle.EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1125) Google Scholar). The mechanisms of SAMHD1 regulation in dividing cells or how Thr592 phosphorylation is modulated remain not fully understood. We aimed to better understand the molecular mechanism of SAMHD1 phosphorylation in dividing cells. In this study, we identified a cyclin-binding motif in SAMHD1 and further analyzed the structural basis underlying the mechanism of the cyclin A2-CDK complex interaction with SAMHD1. Our data on SAMHD1 RXL motif and its interaction with cyclin A suggest effects on SAMHD1 protein expression, phosphorylation, localization, protein stability, and tetramerization of SAMHD1. We have previously shown that overexpressed SAMHD1 interacts with cyclin A1, CDK1, and CDK2 in HEK293T cells (29.St. Gelais C. de Silva S. Hach J.C. White T.E. Diaz-Griffero F. Yount J.S. Wu L. Identification of cellular proteins interacting with the retroviral restriction factor SAMHD1.J. Virol. 2014; 88: 5834-5844Crossref PubMed Scopus (74) Google Scholar). However, the validity of these interactions at physiological expression levels in cells is not known. To determine whether endogenous SAMHD1 interacts with cyclin A, CDK1, and CDK2, we utilized a THP-1 cell line that expresses endogenous SAMHD1 at a level comparable with primary monocyte-derived macrophages (MDM). Using a co-immunoprecipitation (co-IP) approach, we first immunoprecipitated SAMHD1 and confirmed enrichment of SAMHD1 in the IP products (Fig. 1A). Next, we probed for interacting proteins using antibodies to cyclin A, CDK1, and CDK2 and found that cyclin A interacted clearly with SAMHD1, whereas CDK1 and CDK2 showed a weaker interaction. A mouse IgG negative control indicated low levels of nonspecific binding (Fig. 1A). To further validate these interactions, we performed reverse IP for cyclin A, CDK1, and CDK2 and confirmed a strong interaction between cyclin A and SAMHD1. Consistent with Fig. 1A, CDK1 and CDK2 showed a weak interaction with SAMHD1 (Fig. 1B). To visualize the co-localization of SAMHD1 with cyclin and CDK1/2, we performed immunofluorescence analysis in THP-1 cells and MDM. We observed nuclear localization of SAMHD1, with some cytoplasmic puncta visible in THP-1 cells (Fig. 1C) and MDM (Fig. 1D). As expected, cyclin A showed predominantly nuclear staining with some diffuse staining in the cytoplasm. Analysis of the SAMHD1 and cyclin A signals confirmed co-localization of the two proteins within the nucleus of THP-1 cells and MDMs (Fig. 1, C and D). Quantitation of co-localization demonstrated strong co-localization of SAMHD1 with cyclin A in both THP-1 cells and MDMs (Mander's coefficient of 0.88 ± 0.02 and 0.87 ± 0.06, respectively). Similarly, we observed clear co-localization of endogenous SAMHD1 with CDK1/CDK2 within the nucleus of THP-1 cells and MDM (Fig. 1, C and D). Similar values of SAMHD1/CDK2 signal were noted in MDMs (Mander's coefficient of 0.93 ± 0.02, whereas co-localization of SAMHD1/CDK1 was somewhat less (Mander's coefficient of 0.81 ± 0.04, MDM). Together, the co-IP and localization data suggest that endogenous SAMHD1 interacts with cyclin A, CDK1, and CDK2 in cells. Previous publications have demonstrated that SAMHD1 is a substrate of CDK1/2, mediated through a CDK motif located in the C-terminal region of SAMHD1 at residues 592–595 (16.White T.E. Brandariz-Nuñez A. Valle-Casuso J.C. Amie S. Nguyen L. Kim B. Brojatsch J. Diaz-Griffero F. Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1.Virology. 2013; 436: 81-90Crossref PubMed Scopus (103) Google Scholar) (Fig. 2A). We hypothesized that SAMHD1 phosphorylation occurs through interaction with cyclin A via a putative cyclin-binding motif. Sequence alignment of SAMHD1 to proteins that contain a consensus cyclin-binding motif (RXL) highlighted a sequence of conserved residues in SAMHD1 that are similar to proteins containing cyclin-CDK substrate recognition motifs (Fig. 2B) (43.Wohlschlegel J.A. Dwyer B.T. Takeda D.Y. Dutta A. Mutational analysis of the Cy motif from p21 reveals sequence degeneracy and specificity for different cyclin-dependent kinases.Mol. Cell. Biol. 2001; 21: 4868-4874Crossref PubMed Scopus (67) Google Scholar, 45.Adams P.D. Sellers W.R. Sharma S.K. Wu A.D. Nalin C.M. Kaelin Jr., W.G. Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors.Mol. Cell. Biol. 1996; 16: 6623-6633Crossref PubMed Scopus (316) Google Scholar). To understand the structural basis of SAMHD1 binding to cyclin A-CDK complexes, we generated a panel of mutants targeting the conserved Arg451, Leu453, and Phe454 residues (Fig. 2B), which we predicted would affect cyclin A binding to SAMHD1, prevent recruitment of the CDK1/2 to the C-terminal CDK motif, and thus negatively affect SAMHD1 phosphorylation at Thr592. We overexpressed mutant and WT SAMHD1 in HEK293T cells and immunoblotted HA-tagged exogenous SAMHD1 and Thr(P)592 SAMHD1. Interestingly, we found that theRL → AA, RL → KV, and RLF → AAA mutants expressed at lower levels compared with the WT protein (Fig. 2C), suggesting either reduced protein stability or increased degradation. When the plasmid DNA input of mutants was increased 10-fold to compensate for low expression levels, we were able to express the mutant proteins to levels near that of the WT protein. However, the phosphorylation status of the mutants remained low compared with the WT SAMHD1 (Fig. 2C). These results suggest that the RXL motif is likely important for Thr592 phosphorylation of SAMHD1. Previous data has shown that a R451E mutant affected SAMHD1 tetramerization and in vitro dNTPase activity (46.Zhu C. Gao W. Zhao K. Qin X. Zhang Y. Peng X. Zhang L. Dong Y. Zhang W. Li P. Wei W. Gong Y. Yu X.F. Structural insight into dGTP-dependent activation of tetrameric SAMHD1 deoxynucleoside triphosphate triphosphohydrolase.Nat. Commun. 2013; 4: 2722Crossref PubMed Scopus (89) Google Scholar) and that this residue could be involved in binding nucleotides in the allosteric site (32.Arnold L.H. Groom H.C. Kunzelmann S. Schwefel D. Caswell S.J. Ordonez P. Mann M.C. Rueschenbaum S. Goldstone D.C. Pennell S. Howell S.A. Stoye J.P. Webb M. Taylor I.A. Bishop K.N. Phospho-dependent regulation of SAMHD1 oligomerisation couples catalysis and restriction.PLoS Pathog. 2015; 11: e1005194Crossref PubMed Scopus (76) Google Scholar). Although the RXL residues are predicted to be the principal conserved residues involved in cyclin binding, it is possible that adjoining residues could have roles in mediating cyclin binding or specificity (45
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