Title: Skp’n with Cks1: Revelations from the Skp1-Skp2-Cks1-p27 Structure
Abstract: The elucidation of the Skp2 structure complexed with its cofactor Cks1 and bound to a phosphopeptide corresponding to the phosphodegron of the SCFSkp2 substrate p27Kip1 (Hao et al., 2005Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20 (this issue): 9-19Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, this issue of Molecular Cell) resolves several key structure-function issues but, intriguingly, raises a number of new questions to ponder. The elucidation of the Skp2 structure complexed with its cofactor Cks1 and bound to a phosphopeptide corresponding to the phosphodegron of the SCFSkp2 substrate p27Kip1 (Hao et al., 2005Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20 (this issue): 9-19Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, this issue of Molecular Cell) resolves several key structure-function issues but, intriguingly, raises a number of new questions to ponder. Often it is the unexpected findings that ultimately provide the greatest conceptual advances. Several years ago, investigations of Cks1, a high-affinity cyclin-dependent kinase (Cdk) binding protein, and Skp2, the F box protein and specificity factor of the protein ubiquitin ligase, SCFSkp2, experienced a surprising convergence. Two groups, one carrying out mouse genetic experiments to determine the function of Cks1 (Spruck et al., 2001Spruck C. Strohmaier H. Watson M. Smith A.P.L. Ryan A. Krek W. Reed S.I. Mol. Cell. 2001; 7: 639-650Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) and the other pursuing a biochemical strategy to elucidate the requirements for in vitro reconstitution of ubiquitylation of the Cdk inhibitor p27Kip1 (Ganoth et al., 2001Ganoth D. Bornstein G. Ko T.K. Larsen B. Tyers M. Pagano M. Hershko A. Nat. Cell Biol. 2001; 3: 321-324Crossref PubMed Scopus (409) Google Scholar), simultaneously came to the conclusion that Cks1 was an essential cofactor for targeting SCFSkp2 to phosphorylated p27. This was unexpected for several reasons. First, although the function or functions of Cks proteins were not known and remain quite obscure, it was presumed that regulation of Cdk activity was likely at the core. Yet a Cks1 mutant incapable of binding Cdks could provide the essential function, which was also completely resistant to Cdk inhibition (Spruck et al., 2001Spruck C. Strohmaier H. Watson M. Smith A.P.L. Ryan A. Krek W. Reed S.I. Mol. Cell. 2001; 7: 639-650Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Secondly, Cks1 is a very small, flat protein composed primarily of a series of four β strands (Arvai et al., 1995Arvai A.S. Bourne Y. Hickey M.J. Tainer J.A. J. Mol. Biol. 1995; 249: 835-842Crossref PubMed Scopus (67) Google Scholar), and it was difficult to envision what it could contribute to promote binding of the much larger and more complex Skp2 leucine-rich repeat (LRR) surface to p27. In the current issue of Molecular Cell, Hao and coworkers directly answer the question of how Cks1 contributes to Skp2 function (Hao et al., 2005Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20 (this issue): 9-19Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The structure reveals that Cks1 is not simply a facilitator but itself actually constitutes a major part of the substrate binding surface that contacts phosphorylated p27. Although the substrate binding domain of Skp2 forms a concave surface comprising ten LRRs, certainly a respectable potential protein-protein interaction surface with sufficient complexity in theory to recruit SCF substrates, this is clearly not the way the system operates. In fact, there are several surprises. First, Skp2 contains a 39 amino acid tail carboxy-terminal to the LRRs that a previous structure determination of the Skp1-Skp2 heterodimer had placed folded back and occupying much of the LRR pocket (Schulman et al., 2000Schulman B.A. Carrano A.C. Jeffrey P.D. Bowen Z. Kinnucan E.R. Finnin M.S. Elledge S.J. Harper J.W. Pagano M. Pavletich N.P. Nature. 2000; 408: 381-386Crossref PubMed Scopus (469) Google Scholar). It had been speculated that a conformational change removing the tail might be required for substrate occupancy. However, in the current structure including Cks1 and substrate phosphopeptide, the position of the tail remains unchanged. In fact, the Skp2 tail, by packing into the concave LRR surface, serves as a platform for binding the relatively flat Cks1 molecule. Therefore, Cks1 makes extensive contacts with both the LRRs and the tail. Furthermore, there are no significant structural changes imparted to Cks1 or Skp2 as a result of the formation of this complex: in effect, the rigid sum of preformed components creates a combinatorial substrate binding site. The second surprise is the degree to which Cks1 participates in substrate binding. The phosphate corresponding to the activated phosphodegron of p27 (pThr187) binds exclusively to Cks1 in an anion binding pocket predicted from previous Cks protein crystal structures (Arvai et al., 1995Arvai A.S. Bourne Y. Hickey M.J. Tainer J.A. J. Mol. Biol. 1995; 249: 835-842Crossref PubMed Scopus (67) Google Scholar, Parge et al., 1993Parge H.E. Arvai A.S. Murtari D.J. Reed S.I. Tainer J.A. Science. 1993; 262: 387-395Crossref PubMed Scopus (108) Google Scholar) where the site is always occupied by a phosphate or sulfate. The other key residue for substrate interaction is Glu185, which fits into a groove between Cks1 and Skp2 and interacts with residues from both proteins. There are few detectable contacts with other p27 side chains, a finding supported by biochemical data (Hao et al., 2005Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20 (this issue): 9-19Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Therefore, the majority of contacts between substrate and SCFSkp2 are actually with Cks1. This raises the third surprising revelation, and that is the minimal level of complexity and therefore intrinsic specificity of the Skp2-Cks1 phosphodegron consensus. In effect, only two substrate residues make strong contacts with the binding surface: Glu185 and pThr187. Inspection of other known Skp2 targets, p57Kip2 (Kamura et al., 2003Kamura T. Hara T. Kotoshiba S. Yada M. Ishida N. Imaki H. Hatakeyama S. Nakayama K. Nakayama K.I. Proc. Natl. Acad. Sci. USA. 2003; 100: 10231-10236Crossref PubMed Scopus (239) Google Scholar) and p21Cip1 (Bornstein et al., 2003Bornstein G. Bloom J. Sitry-Shevah D. Nakayama K. Pagano M. Hershko A. J. Biol. Chem. 2003; 278: 25752-25757Crossref PubMed Scopus (375) Google Scholar), reinforce this view, as each has a glutamate at position −2 relative to the phosphorylated residue. However, the p21 phosphodegron contains phosphoserine rather than phosphothreonine (Bornstein et al., 2003Bornstein G. Bloom J. Sitry-Shevah D. Nakayama K. Pagano M. Hershko A. J. Biol. Chem. 2003; 278: 25752-25757Crossref PubMed Scopus (375) Google Scholar), indicating an even greater degree of degeneracy. How, then, is substrate specificity achieved? One clue comes from the structure of Cks1 bound to Cdk2 (modeled with cyclin A also bound) (Bourne et al., 1996Bourne Y. Watson M.H. Hickey M.J. Holmes W. Rocque W. Reed S.I. Tainer J.A. Cell. 1996; 84: 863-874Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, Jeffrey et al., 1995Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massague J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1172) Google Scholar). Based on this and the current structure, it is predicted that Cks1 can bind Skp2, p27, Cdk2, and cyclin A simultaneously. Since Skp2 binds independently to Cdk2 and cyclin A (Yam et al., 1999Yam C.H. Ng R.W. Siu W.Y. Lau A.W. Poon R.Y. Mol. Cell. Biol. 1999; 19: 635-645Crossref PubMed Scopus (71) Google Scholar) and all three SCFSkp2 targets cited above are presented for ubiquitylation as Cdk-cyclin-inhibitor heterotrimers, the binding between Skp2, Cdk, and cyclin would be expected to add significant affinity and interaction specificity. Indeed, a Cks1 mutant incapable of binding to Cdks, although functional (Spruck et al., 2001Spruck C. Strohmaier H. Watson M. Smith A.P.L. Ryan A. Krek W. Reed S.I. Mol. Cell. 2001; 7: 639-650Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar), shows reduced efficiency in vitro, particularly under limiting conditions (Sitry et al., 2002Sitry D. Seeliger M.A. Ko T.K. Ganoth D. Breward S.E. Itzhaki L.S. Pagano M. Hershko A. J. Biol. Chem. 2002; 277: 42233-42240Crossref PubMed Scopus (76) Google Scholar). In addition to answering many questions related to Skp2 function, the Skp2-Cks1 structure raises numerous others. What is the relevance of this structure to other LRR-containing F box proteins (Fbxls) that presumably serve as specificity factors for SCF ubiquitin ligases? What does the role of Cks1 in Skp2-substrate binding speak to the other functions of Cks proteins? Hao and coworkers (Hao et al., 2005Hao B. Zheng N. Schulman B.A. Wu G. Miller J.J. Pagano M. Pavletich N.P. Mol. Cell. 2005; 20 (this issue): 9-19Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) address the first question by determining whether any other Fbxls can bind Cks1. They test this theoretically and empirically, and the answer in both instances is “no.” The other Fbxls exhibit no homology between their C-terminal tails and that of Skp2, and, in pull-down experiments, Cks1 only binds to Skp2. However, comparison of tail sequences between orthologs expressed in different species indicates that tails can be highly conserved in evolution, suggesting a specific protein interaction role. Similarly, orthologous sequence conservation specifically within a subdomain of the LRR region that corresponds to the Cks1-substrate binding surface of Skp2 is consistent with a conserved role in protein-protein interaction. Therefore, it is likely that many if not most Fbxls use a combination of C-terminal tail and LRR structures to recruit targets to SCF complexes. Whether these Fbxls also utilize a protein cofactor such as Cks1 to build a binding site remains to be determined. The role of Cks1 as a phosphate binding module in Skp2-target interactions suggests that Cks proteins may carry out similar functions in other contexts. Although many of the functions of Cks proteins are likely not yet known, it has been suggested that Cks proteins may help target Cdk-cyclin complexes, to which they bind with high affinity, to Cdk substrates (Patra et al., 1999Patra D. Wang S.X. Kumagai A. Dunphy W.G. J. Biol. Chem. 1999; 274: 36839-36842Crossref PubMed Scopus (91) Google Scholar). Perhaps the ability of Cks proteins to bind phosphorylated residues allows regulation or priming of Cdk-substrate interactions via prior phosphorylation by a second kinase. In yeast, Cks1 is required for recruiting proteasomes to chromatin in order to potentiate transcription of a subset of genes (Yu et al., 2005Yu V.P. Baskerville C. Grunenfelder B. Reed S.I. Mol. Cell. 2005; 17: 145-151Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). This function may be more akin to that elucidated for Skp2-substrate binding in that, although Cdk1 is required for binding of Cks1 to chromatin, the kinase activity of Cdk1 is not. Therefore, as with Skp2, interactions between Cdk1 and unknown chromatin proteins may serve to shore up a relatively weak and low-specificity interaction between Cks1 and a minimal phosphorylated consensus on a putative receptor. Although Cks proteins have remained elusive and mysterious for 20 years, the structure of the Skp1-Skp2-Cks1-p27 complex is certain to redefine thinking about how to approach their function(s).