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Identification of a Tankyrase-binding Motif Shared by IRAP, TAB182, and Human TRF1 but Not Mouse TRF1

2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês

10.1074/jbc.m203916200

ISSN

1083-351X

Autores

Juan I. Sbodio, Nai-Wen Chi,

Tópico(s)

Microtubule and mitosis dynamics

Resumo

Tankyrase-1 and -2 are closely related poly(ADP-ribose) polymerases that use an ankyrin-repeat domain to bind diverse proteins, including TRF (telomere-repeat binding factor)-1, IRAP (insulin-responsiveaminopeptidase), and TAB182 (182-kDatankyrase-binding protein). TRF1 binding allows tankyrase to regulate telomere dynamics in human cells, whereas IRAP binding presumably allows tankyrase to regulate the targeting of IRAP. The mechanism by which tankyrase binds to diverse proteins has not been investigated. Herein we describe a novel RXXPDG motif shared by IRAP, TAB182, and human TRF1 that mediates their binding to tankyrases. Interestingly, mouse TRF1 lacks this motif and thus does not bind either tankyrase-1 or -2. Using the ankyrin domain of tankyrase as a bait in a yeast two-hybrid screen, we also found the RXXPDG motif in six candidate tankyrase partners, including the nuclear/mitotic apparatus protein (NuMA). We verified NuMA as an RXXPDG-mediated partner of tankyrase and suggest that this interaction contributes to the known colocalization of tankyrase and NuMA at mitotic spindle poles. Tankyrase-1 and -2 are closely related poly(ADP-ribose) polymerases that use an ankyrin-repeat domain to bind diverse proteins, including TRF (telomere-repeat binding factor)-1, IRAP (insulin-responsiveaminopeptidase), and TAB182 (182-kDatankyrase-binding protein). TRF1 binding allows tankyrase to regulate telomere dynamics in human cells, whereas IRAP binding presumably allows tankyrase to regulate the targeting of IRAP. The mechanism by which tankyrase binds to diverse proteins has not been investigated. Herein we describe a novel RXXPDG motif shared by IRAP, TAB182, and human TRF1 that mediates their binding to tankyrases. Interestingly, mouse TRF1 lacks this motif and thus does not bind either tankyrase-1 or -2. Using the ankyrin domain of tankyrase as a bait in a yeast two-hybrid screen, we also found the RXXPDG motif in six candidate tankyrase partners, including the nuclear/mitotic apparatus protein (NuMA). We verified NuMA as an RXXPDG-mediated partner of tankyrase and suggest that this interaction contributes to the known colocalization of tankyrase and NuMA at mitotic spindle poles. poly(ADP-ribose) polymerase ankyrin repeat telomeric-repeat binding factor insulin-responsive aminopeptidase mitogen-activated protein 182-kDa tankyrase-binding protein nuclear/mitotic apparatus protein ANK-repeat clusters amino acid(s) glutathione S-transferase Tankyrase-1 is a modular protein with both enzymatic and scaffolding activities (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Its PARP1 domain catalyzes thepoly(ADP-ribosyl)ation of substrateproteins, whereas its ankyrin (ANK)-repeat domain interacts with diverse partners, apparently serving to recruit substrates for the PARP domain. Thus, tankyrase-1 binds to thetelomeric-repeat binding factor TRF1 and poly(ADP-ribosyl)ates it (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Because TRF1 opposes telomere elongation in vivo and its affinity for telomeric DNA is diminished by poly(ADP-ribosyl)ation in vitro, tankyrase-1 has been proposed to regulate telomere dynamics by antagonizing TRF1 (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Indeed, telomeres progressively expand in human cell lines stably overexpressing tankyrase-1 tagged with a nuclear localization sequence (2Smith S. de Lange T. Curr. Biol. 2000; 10: 1299-1302Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Besides binding to TRF1, the ANK domain of tankyrase-1 also binds to an angiotensin-4 receptor previously known as IRAP (insulin-responsiveaminopeptidase) (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 4Albiston A.L. McDowall S.G. Matsacos D. Sim P. Clune E. Mustafa T. Lee J. Mendelsohn F.A. Simpson R.J. Connolly L.M. Chai S.Y. J. Biol. Chem. 2001; 276: 48623-48626Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). IRAP is a recycling vesicular protein that is recruited to the cell surface by insulin signaling. IRAP binding to tankyrase-1 has led to the speculation that tankyrase-1, particularly given its PARP activity, regulates IRAP targeting in response to insulin (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). This is because tankyrase-1 is phosphorylated by mitogen-activated protein kinases in insulin-stimulated cells, and this phosphorylation enhances the PARP activity of tankyrase-1 (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). A third protein known to bind the ANK domain of tankyrase-1 is a novel 182-kDatankyrase-binding protein called TAB182, whose function is largely unknown except for its dual targeting to both the nucleus and the periphery of cytosol (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar).Functional characterization of tankyrase-1 has been confounded by its complex targeting pattern that varies throughout the cell cycle (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). During mitosis, tankyrase-1 is concentrated around the centrosomes at either end of the bipolar mitotic apparatus (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). Among several centrosomal markers tested, the spindle pole marker NuMA (nuclear/mitotic apparatus protein) colocalizes best with tankyrase-1 (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). This colocalization ceases after mitosis, when NuMA returns to the nucleus (7Zeng C. Microsc. Res. Tech. 2000; 49: 467-477Crossref PubMed Scopus (60) Google Scholar) while tankyrase-1 becomes associated with Golgi membranes that coalesce around the centrosomes (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Although Golgi and spindle poles alternately contain the bulk of tankyrase-1, a small fraction of tankyrase-1 is targeted to human telomeres through TRF1 binding (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar, 6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). For yet-unclear reasons, mouse telomeres do not contain detectable amounts of tankyrase-1 by immunofluorescence analysis (8Scherthan H. Jerratsch M., Li, B. Smith S. Hulten M. Lock T. de Lange T. Mol. Biol. Cell. 2000; 11: 4189-4203Crossref PubMed Scopus (127) Google Scholar).Tankyrase-1 is mirrored in many aspects by its closely related homologue, tankyrase-2 (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar, 10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Kaminker P.G. Kim S.H. Taylor R.D. Zebarjadian Y. Funk W.D. Morin G.B. Yaswen P. Campisi J. J. Biol. Chem. 2001; 276: 35891-35899Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In fact, both tankyrases hetero-oligomerize and colocalize in vivo (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar). Like tankyrase-1, tankyrase-2 is proposed to regulate protein targeting in response to growth factor signaling (10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and tankyrase-2 overexpression (tagged with a nuclear localization sequence) in human cells has been shown to elongate telomeres (12Cook B.D. Dynek J.N. Chang W. Shostak G. Smith S. Mol. Cell. Biol. 2002; 22: 332-342Crossref PubMed Scopus (247) Google Scholar). Moreover, the ANK domain of tankyrase-2 also binds to TRF1, IRAP, and TAB182 (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar,11Kaminker P.G. Kim S.H. Taylor R.D. Zebarjadian Y. Funk W.D. Morin G.B. Yaswen P. Campisi J. J. Biol. Chem. 2001; 276: 35891-35899Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar).The specific binding of these three apparently unrelated proteins to tankyrases suggests two likely scenarios. First, a cryptic structural motif common to TRF1, IRAP, and TAB182 could enable them to bind tankyrase using the same mechanism. Alternatively, each partner might use a unique mechanism to bind distinct sites within the ANK domain of tankyrase. According to our sequence analysis, the ANK domain comprises 20 ANK repeats (19 full repeats plus two flanking half-repeats) that are evenly demarcated into five ANK subdomains by a recurrent insertional sequence (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar) (Fig. 3, herein). Consistent with these subdomains arising through gene quintuplication, the full blown ANK domain contains redundant binding sites for IRAP (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar). In an alternative sequence analysis that invokes multiple irregular insertions and deletions, the ANK domain is depicted as comprising 24, instead of 20, ANK repeats that are grouped into fiveANK-repeat clusters or ARCs (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar, 5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These ARCs generally correspond to the five ANK subdomains that we defined (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar), and each ARC represents an independent binding site in tankyrase (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Importantly, a yeast two-hybrid analysis showed that ARC-I and -V interact with TRF1 but not with TAB182, whereas the other three ARCs interact with both partners (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Such a partner selectivity by ARCs (hereafter referred to as ANK subdomains) suggests a mechanistic distinction in how TRF1 and TAB182 bind to tankyrase. However, biochemical analyses herein indicate that ANK subdomain-V of tankyrase binds indiscriminately to TRF1, IRAP, and TAB182. Moreover, all three partners use a shared RXXPDG motif to bind tankyrase. This tankyrase-binding motif prompted us to uncover NuMA as an RXXPDG-containing partner of tankyrase. We also show that mouse TRF1, unlike human TRF1, lacks an RXXPDG motif in its acidic domain and thus does not bind to tankyrase. Therefore, the RXXPDG motif is an important determinant for diverse proteins to bind the ANK domain of tankyrase.DISCUSSIONThis study describes an RXXPDG motif in IRAP, TAB182, and human TRF1 that binds the ANK domain of tankyrase. Our yeast two-hybrid screen also revealed this RXXPDG motif in additional candidate interactors of tankyrase. Moreover, we established NuMA as an RXXPDG-containing partner of tankyrase.ANK repeats form the protein-protein interacting domain of numerous molecules; at least four repeats are required to form an ANK domain (22Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). The ANK domains in the p53-binding protein 53BP2 and in the cytoskeletal protein ankyrin are known to bind to diverse partners. More specifically, 53BP2 binds to p53, Bcl2, and the catalytic subunit of the protein phosphatase PP1 (23Naumovski L. Cleary M.L. Mol. Cell. Biol. 1996; 16: 3884-3892Crossref PubMed Google Scholar, 24Helps N.R. Barker H.M. Elledge S.J. Cohen P.T. FEBS Lett. 1995; 377: 295-300Crossref PubMed Scopus (125) Google Scholar), whereas ankyrin binds to at least seven distinct membrane proteins (referenced in Ref. 25Michaely P. Bennett V. J. Biol. Chem. 1995; 270: 31298-31302Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, neither 53BP2 nor ankyrin has a known binding motif that unifies its diverse partners. Therefore, tankyrase is unique in having a defined binding motif that is critical for binding IRAP, TAB182, and hTRF1 (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (Fig. 2). In IRAP and TAB182, a hexapeptide sequence matching the motif is sufficient by itself for efficient tankyrase binding (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (Fig. 2A). By contrast, hTRF1 contains a variant RXXPDG hexapeptide that requires flanking sequences for efficient tankyrase binding (Fig. 2B).Our biochemical analysis showed that IRAP, TAB182, and hTRF1 all can interact with a minimal binding site (subdomain V) in tankyrase, consistent with their using the same RXXPDG-dependent binding mechanism (Fig. 3). This finding contradicts a previous yeast two-hybrid assay where subdomain V of tankyrase interacts with hTRF1 but not with TAB182 (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Of note, when assayed in yeasts for the interaction with various tankyrase regions, hTRF1 systematically scores higher than TAB182 (up to 16-fold), but its interaction with subdomain V is relatively weak (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It is therefore not surprising that the yeast system fails to detect the TAB182 interaction of subdomain V.The specificity of human tankyrases for RXXPDG is apparently conserved by mouse tankyrases, thus explaining the inability of the latter to bind the RXXPDG-less mTRF1 (Fig. 6). Because telomeric targeting of human tankyrase requires TRF1 binding (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar), lack of this binding explains the absence of detectable tankyrase at mouse telomeres (8Scherthan H. Jerratsch M., Li, B. Smith S. Hulten M. Lock T. de Lange T. Mol. Biol. Cell. 2000; 11: 4189-4203Crossref PubMed Scopus (127) Google Scholar) and implies that tankyrase does not regulate telomeres in mice as it does in humans.The RXXPDG motif is not always an accurate predictor for tankyrase binding, because its residues 2 and 3 are not entirely neutral, as evidenced by the failure of the RXXPDG hexapeptide REYPDG to bind to tankyrase (data not shown). Moreover, the context of a motif may preclude its interaction with tankyrase. For example, the hexapeptideRDTPDG by itself can bind tankyrase, but an SH2 domain containing this hexapeptide cannot bind (GenBankTMCAA56868; data not shown). As another caveat, Grb14 (an adapter for growth factor receptors) lacks a recognizable RXXPDG motif but nevertheless binds to the ANK domain of tankyrase-2 (10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In this unusual case, it remains to be shown if Grb14 uses a degenerative RXXPDG sequence (such as its LPLPDG56 hexapeptide) or an unrelated mechanism to bind tankyrase. Despite these caveats, RXXPDG is clearly important in allowing tankyrase to bind many partners, including NuMA (Fig. 4).The significance of tankyrase binding to NuMA remains unknown. Given their mitotic colocalization at spindle poles (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar), we propose that tankyrase acts as a spindle-pole scaffold that recruits NuMA upon its mitotic release from the nucleus. This notion is supported by tankyrase binding to the polar targeting domain of NuMA (Fig. 4) (7Zeng C. Microsc. Res. Tech. 2000; 49: 467-477Crossref PubMed Scopus (60) Google Scholar). Moreover, the RXXPDG sequence of NuMA (RTQPDG1748, Fig. 4D) resides in a small region (aa 1667–1766) that, when deleted, impairs the polar targeting of NuMA (Ref. 26Tang T.K. Tang C.J. Chao Y.J. Wu C.W. J. Cell Sci. 1994; 107: 1389-1402PubMed Google Scholar but also see Ref. 27Gueth-Hallonet C. Weber K. Osborn M. Exp. Cell Res. 1996; 225: 207-218Crossref PubMed Scopus (32) Google Scholar). Tankyrase-1 is a modular protein with both enzymatic and scaffolding activities (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Its PARP1 domain catalyzes thepoly(ADP-ribosyl)ation of substrateproteins, whereas its ankyrin (ANK)-repeat domain interacts with diverse partners, apparently serving to recruit substrates for the PARP domain. Thus, tankyrase-1 binds to thetelomeric-repeat binding factor TRF1 and poly(ADP-ribosyl)ates it (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Because TRF1 opposes telomere elongation in vivo and its affinity for telomeric DNA is diminished by poly(ADP-ribosyl)ation in vitro, tankyrase-1 has been proposed to regulate telomere dynamics by antagonizing TRF1 (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Indeed, telomeres progressively expand in human cell lines stably overexpressing tankyrase-1 tagged with a nuclear localization sequence (2Smith S. de Lange T. Curr. Biol. 2000; 10: 1299-1302Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Besides binding to TRF1, the ANK domain of tankyrase-1 also binds to an angiotensin-4 receptor previously known as IRAP (insulin-responsiveaminopeptidase) (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 4Albiston A.L. McDowall S.G. Matsacos D. Sim P. Clune E. Mustafa T. Lee J. Mendelsohn F.A. Simpson R.J. Connolly L.M. Chai S.Y. J. Biol. Chem. 2001; 276: 48623-48626Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). IRAP is a recycling vesicular protein that is recruited to the cell surface by insulin signaling. IRAP binding to tankyrase-1 has led to the speculation that tankyrase-1, particularly given its PARP activity, regulates IRAP targeting in response to insulin (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). This is because tankyrase-1 is phosphorylated by mitogen-activated protein kinases in insulin-stimulated cells, and this phosphorylation enhances the PARP activity of tankyrase-1 (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). A third protein known to bind the ANK domain of tankyrase-1 is a novel 182-kDatankyrase-binding protein called TAB182, whose function is largely unknown except for its dual targeting to both the nucleus and the periphery of cytosol (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Functional characterization of tankyrase-1 has been confounded by its complex targeting pattern that varies throughout the cell cycle (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). During mitosis, tankyrase-1 is concentrated around the centrosomes at either end of the bipolar mitotic apparatus (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). Among several centrosomal markers tested, the spindle pole marker NuMA (nuclear/mitotic apparatus protein) colocalizes best with tankyrase-1 (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). This colocalization ceases after mitosis, when NuMA returns to the nucleus (7Zeng C. Microsc. Res. Tech. 2000; 49: 467-477Crossref PubMed Scopus (60) Google Scholar) while tankyrase-1 becomes associated with Golgi membranes that coalesce around the centrosomes (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Although Golgi and spindle poles alternately contain the bulk of tankyrase-1, a small fraction of tankyrase-1 is targeted to human telomeres through TRF1 binding (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar, 6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar). For yet-unclear reasons, mouse telomeres do not contain detectable amounts of tankyrase-1 by immunofluorescence analysis (8Scherthan H. Jerratsch M., Li, B. Smith S. Hulten M. Lock T. de Lange T. Mol. Biol. Cell. 2000; 11: 4189-4203Crossref PubMed Scopus (127) Google Scholar). Tankyrase-1 is mirrored in many aspects by its closely related homologue, tankyrase-2 (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar, 10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Kaminker P.G. Kim S.H. Taylor R.D. Zebarjadian Y. Funk W.D. Morin G.B. Yaswen P. Campisi J. J. Biol. Chem. 2001; 276: 35891-35899Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In fact, both tankyrases hetero-oligomerize and colocalize in vivo (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar). Like tankyrase-1, tankyrase-2 is proposed to regulate protein targeting in response to growth factor signaling (10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), and tankyrase-2 overexpression (tagged with a nuclear localization sequence) in human cells has been shown to elongate telomeres (12Cook B.D. Dynek J.N. Chang W. Shostak G. Smith S. Mol. Cell. Biol. 2002; 22: 332-342Crossref PubMed Scopus (247) Google Scholar). Moreover, the ANK domain of tankyrase-2 also binds to TRF1, IRAP, and TAB182 (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar,11Kaminker P.G. Kim S.H. Taylor R.D. Zebarjadian Y. Funk W.D. Morin G.B. Yaswen P. Campisi J. J. Biol. Chem. 2001; 276: 35891-35899Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The specific binding of these three apparently unrelated proteins to tankyrases suggests two likely scenarios. First, a cryptic structural motif common to TRF1, IRAP, and TAB182 could enable them to bind tankyrase using the same mechanism. Alternatively, each partner might use a unique mechanism to bind distinct sites within the ANK domain of tankyrase. According to our sequence analysis, the ANK domain comprises 20 ANK repeats (19 full repeats plus two flanking half-repeats) that are evenly demarcated into five ANK subdomains by a recurrent insertional sequence (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar) (Fig. 3, herein). Consistent with these subdomains arising through gene quintuplication, the full blown ANK domain contains redundant binding sites for IRAP (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar). In an alternative sequence analysis that invokes multiple irregular insertions and deletions, the ANK domain is depicted as comprising 24, instead of 20, ANK repeats that are grouped into fiveANK-repeat clusters or ARCs (1Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar, 5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). These ARCs generally correspond to the five ANK subdomains that we defined (9Sbodio J.I. Lodish H.F. Chi N.-W. Biochem. J. 2002; 361: 451-459Crossref PubMed Scopus (108) Google Scholar), and each ARC represents an independent binding site in tankyrase (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Importantly, a yeast two-hybrid analysis showed that ARC-I and -V interact with TRF1 but not with TAB182, whereas the other three ARCs interact with both partners (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Such a partner selectivity by ARCs (hereafter referred to as ANK subdomains) suggests a mechanistic distinction in how TRF1 and TAB182 bind to tankyrase. However, biochemical analyses herein indicate that ANK subdomain-V of tankyrase binds indiscriminately to TRF1, IRAP, and TAB182. Moreover, all three partners use a shared RXXPDG motif to bind tankyrase. This tankyrase-binding motif prompted us to uncover NuMA as an RXXPDG-containing partner of tankyrase. We also show that mouse TRF1, unlike human TRF1, lacks an RXXPDG motif in its acidic domain and thus does not bind to tankyrase. Therefore, the RXXPDG motif is an important determinant for diverse proteins to bind the ANK domain of tankyrase. DISCUSSIONThis study describes an RXXPDG motif in IRAP, TAB182, and human TRF1 that binds the ANK domain of tankyrase. Our yeast two-hybrid screen also revealed this RXXPDG motif in additional candidate interactors of tankyrase. Moreover, we established NuMA as an RXXPDG-containing partner of tankyrase.ANK repeats form the protein-protein interacting domain of numerous molecules; at least four repeats are required to form an ANK domain (22Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). The ANK domains in the p53-binding protein 53BP2 and in the cytoskeletal protein ankyrin are known to bind to diverse partners. More specifically, 53BP2 binds to p53, Bcl2, and the catalytic subunit of the protein phosphatase PP1 (23Naumovski L. Cleary M.L. Mol. Cell. Biol. 1996; 16: 3884-3892Crossref PubMed Google Scholar, 24Helps N.R. Barker H.M. Elledge S.J. Cohen P.T. FEBS Lett. 1995; 377: 295-300Crossref PubMed Scopus (125) Google Scholar), whereas ankyrin binds to at least seven distinct membrane proteins (referenced in Ref. 25Michaely P. Bennett V. J. Biol. Chem. 1995; 270: 31298-31302Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, neither 53BP2 nor ankyrin has a known binding motif that unifies its diverse partners. Therefore, tankyrase is unique in having a defined binding motif that is critical for binding IRAP, TAB182, and hTRF1 (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (Fig. 2). In IRAP and TAB182, a hexapeptide sequence matching the motif is sufficient by itself for efficient tankyrase binding (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (Fig. 2A). By contrast, hTRF1 contains a variant RXXPDG hexapeptide that requires flanking sequences for efficient tankyrase binding (Fig. 2B).Our biochemical analysis showed that IRAP, TAB182, and hTRF1 all can interact with a minimal binding site (subdomain V) in tankyrase, consistent with their using the same RXXPDG-dependent binding mechanism (Fig. 3). This finding contradicts a previous yeast two-hybrid assay where subdomain V of tankyrase interacts with hTRF1 but not with TAB182 (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Of note, when assayed in yeasts for the interaction with various tankyrase regions, hTRF1 systematically scores higher than TAB182 (up to 16-fold), but its interaction with subdomain V is relatively weak (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It is therefore not surprising that the yeast system fails to detect the TAB182 interaction of subdomain V.The specificity of human tankyrases for RXXPDG is apparently conserved by mouse tankyrases, thus explaining the inability of the latter to bind the RXXPDG-less mTRF1 (Fig. 6). Because telomeric targeting of human tankyrase requires TRF1 binding (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar), lack of this binding explains the absence of detectable tankyrase at mouse telomeres (8Scherthan H. Jerratsch M., Li, B. Smith S. Hulten M. Lock T. de Lange T. Mol. Biol. Cell. 2000; 11: 4189-4203Crossref PubMed Scopus (127) Google Scholar) and implies that tankyrase does not regulate telomeres in mice as it does in humans.The RXXPDG motif is not always an accurate predictor for tankyrase binding, because its residues 2 and 3 are not entirely neutral, as evidenced by the failure of the RXXPDG hexapeptide REYPDG to bind to tankyrase (data not shown). Moreover, the context of a motif may preclude its interaction with tankyrase. For example, the hexapeptideRDTPDG by itself can bind tankyrase, but an SH2 domain containing this hexapeptide cannot bind (GenBankTMCAA56868; data not shown). As another caveat, Grb14 (an adapter for growth factor receptors) lacks a recognizable RXXPDG motif but nevertheless binds to the ANK domain of tankyrase-2 (10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In this unusual case, it remains to be shown if Grb14 uses a degenerative RXXPDG sequence (such as its LPLPDG56 hexapeptide) or an unrelated mechanism to bind tankyrase. Despite these caveats, RXXPDG is clearly important in allowing tankyrase to bind many partners, including NuMA (Fig. 4).The significance of tankyrase binding to NuMA remains unknown. Given their mitotic colocalization at spindle poles (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar), we propose that tankyrase acts as a spindle-pole scaffold that recruits NuMA upon its mitotic release from the nucleus. This notion is supported by tankyrase binding to the polar targeting domain of NuMA (Fig. 4) (7Zeng C. Microsc. Res. Tech. 2000; 49: 467-477Crossref PubMed Scopus (60) Google Scholar). Moreover, the RXXPDG sequence of NuMA (RTQPDG1748, Fig. 4D) resides in a small region (aa 1667–1766) that, when deleted, impairs the polar targeting of NuMA (Ref. 26Tang T.K. Tang C.J. Chao Y.J. Wu C.W. J. Cell Sci. 1994; 107: 1389-1402PubMed Google Scholar but also see Ref. 27Gueth-Hallonet C. Weber K. Osborn M. Exp. Cell Res. 1996; 225: 207-218Crossref PubMed Scopus (32) Google Scholar). This study describes an RXXPDG motif in IRAP, TAB182, and human TRF1 that binds the ANK domain of tankyrase. Our yeast two-hybrid screen also revealed this RXXPDG motif in additional candidate interactors of tankyrase. Moreover, we established NuMA as an RXXPDG-containing partner of tankyrase. ANK repeats form the protein-protein interacting domain of numerous molecules; at least four repeats are required to form an ANK domain (22Sedgwick S.G. Smerdon S.J. Trends Biochem. Sci. 1999; 24: 311-316Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). The ANK domains in the p53-binding protein 53BP2 and in the cytoskeletal protein ankyrin are known to bind to diverse partners. More specifically, 53BP2 binds to p53, Bcl2, and the catalytic subunit of the protein phosphatase PP1 (23Naumovski L. Cleary M.L. Mol. Cell. Biol. 1996; 16: 3884-3892Crossref PubMed Google Scholar, 24Helps N.R. Barker H.M. Elledge S.J. Cohen P.T. FEBS Lett. 1995; 377: 295-300Crossref PubMed Scopus (125) Google Scholar), whereas ankyrin binds to at least seven distinct membrane proteins (referenced in Ref. 25Michaely P. Bennett V. J. Biol. Chem. 1995; 270: 31298-31302Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, neither 53BP2 nor ankyrin has a known binding motif that unifies its diverse partners. Therefore, tankyrase is unique in having a defined binding motif that is critical for binding IRAP, TAB182, and hTRF1 (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (Fig. 2). In IRAP and TAB182, a hexapeptide sequence matching the motif is sufficient by itself for efficient tankyrase binding (3Chi N.-W. Lodish H.F. J. Biol. Chem. 2000; 275: 38437-38444Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (Fig. 2A). By contrast, hTRF1 contains a variant RXXPDG hexapeptide that requires flanking sequences for efficient tankyrase binding (Fig. 2B). Our biochemical analysis showed that IRAP, TAB182, and hTRF1 all can interact with a minimal binding site (subdomain V) in tankyrase, consistent with their using the same RXXPDG-dependent binding mechanism (Fig. 3). This finding contradicts a previous yeast two-hybrid assay where subdomain V of tankyrase interacts with hTRF1 but not with TAB182 (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Of note, when assayed in yeasts for the interaction with various tankyrase regions, hTRF1 systematically scores higher than TAB182 (up to 16-fold), but its interaction with subdomain V is relatively weak (5Seimiya H. Smith S. J. Biol. Chem. 2002; 277: 14116-14126Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It is therefore not surprising that the yeast system fails to detect the TAB182 interaction of subdomain V. The specificity of human tankyrases for RXXPDG is apparently conserved by mouse tankyrases, thus explaining the inability of the latter to bind the RXXPDG-less mTRF1 (Fig. 6). Because telomeric targeting of human tankyrase requires TRF1 binding (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar), lack of this binding explains the absence of detectable tankyrase at mouse telomeres (8Scherthan H. Jerratsch M., Li, B. Smith S. Hulten M. Lock T. de Lange T. Mol. Biol. Cell. 2000; 11: 4189-4203Crossref PubMed Scopus (127) Google Scholar) and implies that tankyrase does not regulate telomeres in mice as it does in humans. The RXXPDG motif is not always an accurate predictor for tankyrase binding, because its residues 2 and 3 are not entirely neutral, as evidenced by the failure of the RXXPDG hexapeptide REYPDG to bind to tankyrase (data not shown). Moreover, the context of a motif may preclude its interaction with tankyrase. For example, the hexapeptideRDTPDG by itself can bind tankyrase, but an SH2 domain containing this hexapeptide cannot bind (GenBankTMCAA56868; data not shown). As another caveat, Grb14 (an adapter for growth factor receptors) lacks a recognizable RXXPDG motif but nevertheless binds to the ANK domain of tankyrase-2 (10Lyons R.J. Deane R. Lynch D.K., Ye, Z.S. Sanderson G.M. Eyre H.J. Sutherland G.R. Daly R.J. J. Biol. Chem. 2001; 276: 17172-17180Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). In this unusual case, it remains to be shown if Grb14 uses a degenerative RXXPDG sequence (such as its LPLPDG56 hexapeptide) or an unrelated mechanism to bind tankyrase. Despite these caveats, RXXPDG is clearly important in allowing tankyrase to bind many partners, including NuMA (Fig. 4). The significance of tankyrase binding to NuMA remains unknown. Given their mitotic colocalization at spindle poles (6Smith S. de Lange T. J. Cell Sci. 1999; 112: 3649-3656Crossref PubMed Google Scholar), we propose that tankyrase acts as a spindle-pole scaffold that recruits NuMA upon its mitotic release from the nucleus. This notion is supported by tankyrase binding to the polar targeting domain of NuMA (Fig. 4) (7Zeng C. Microsc. Res. Tech. 2000; 49: 467-477Crossref PubMed Scopus (60) Google Scholar). Moreover, the RXXPDG sequence of NuMA (RTQPDG1748, Fig. 4D) resides in a small region (aa 1667–1766) that, when deleted, impairs the polar targeting of NuMA (Ref. 26Tang T.K. Tang C.J. Chao Y.J. Wu C.W. J. Cell Sci. 1994; 107: 1389-1402PubMed Google Scholar but also see Ref. 27Gueth-Hallonet C. Weber K. Osborn M. Exp. Cell Res. 1996; 225: 207-218Crossref PubMed Scopus (32) Google Scholar). We thank Dr. Joseph Avruch for pEBG and Dr. William Schiemann for insightful comments.

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