The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases

Abstract

Progression through the eukaryotic cell cycle is driven by the orderly activation of cyclin-dependent kinases (CDKs). For activity, CDKs require association with a cyclin and phosphorylation by a separate protein kinase at a conserved threonine residue (T160 in CDK2). Here we present the structure of a complex consisting of phosphorylated CDK2 and cyclin A together with an optimal peptide substrate, HHASPRK. This structure provides an explanation for the specificity of CDK2 towards the proline that follows the phosphorylatable serine of the substrate peptide, and the requirement for the basic residue in the P+3 position of the substrate. We also present the structure of phosphorylated CDK2 plus cyclin A3 in complex with residues 658-668 from the CDK2 substrate p107. These residues include the RXL motif required to target p107 to cyclins. This structure explains the specificity of the RXL motif for cyclins.

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…This result differs from studies of budding yeast cyclins 52 or the Cdk2/ CycA complex and the substrate recognition site on CycA, which was identified to interact with the RxL motif of the substrate Cdc6 about 20 residues downstream of the phosphorylation site 22 . The corresponding surface patch on the first cyclin box repeat of CycT1 is covered both by P-TEFb-activating factors, such as Tat 33,53 , and by inhibitory factors, such as Hexim 30,31 , which do not directly contribute to substrate binding as shown here.…”

“…1c). Although this result may seem trivial, it confirms a high specificity of P-TEFb for the CTD substrate given that three canonical serines are present in each hepta-repeat, two of them as the preferred Ser-Pro recognition motif for Cdk family kinase phosphorylation 22,23 . It also indicates that a continuous phosphorylation signature can be achieved by P-TEFb, leaving no gaps in the modification pattern of the CTD hepta-repeat structure.…”

“…Cyclin-dependent kinases Cdk1 and Cdk2 typically phosphorylate substrates of the consensus type (S/T)Px(K/R) 22,23 . In this motif, the amino group of the lysine residue at position + 3 is involved in ionic interactions with the phospho-threonine moiety within the T-loop activation segment of the kinase 22 .…”

“…In this motif, the amino group of the lysine residue at position + 3 is involved in ionic interactions with the phospho-threonine moiety within the T-loop activation segment of the kinase 22 . Following this recognition principle, phosphorylation of Ser5 by P-TEFb assigns Tyr1 a central role in the S 5 PSY 1 sequence motif.…”

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…This consensus, or substrate recognition sequence, represents yet another level of substrate specificity and has become a means through which different families of kinases are classified [62]. For example, the consensus sequence of the CMGC kinase family member, cyclin-dependent kinase 2 (CDK2) which corresponds to S/T*-P-X-K/R (where * indicates the phosphorylation site and X denotes any amino acid), dictates that CDK2 substrates must contain a basic residue at the P+3 position to accommodate a phospho-Thr residue within the kinase (Thr160) [49,63,64].…”