The yeast cyclin-dependent kinase inhibitor Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain

Abstract

In Saccharomyces cerevisiae, Sic1, an inhibitor of Cdk (cyclindependent kinase), blocks the activity of S-Cdk1 (Cdk1/Clb5,6) kinase that is required for DNA replication. Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase. Despite the well-documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure. In this paper, we show that Sic1 is functionally and structurally related to the mammalian Cki (Cdk inhibitor) p27Kip1 of the Kip/Cip family. A molecular model of the inhibitory domain of Sic1 bound to the Cdk2-cyclin A complex suggested that the yeast inhibitor might productively interface with the mammalian Cdk2-cyclin A complex. Consistent with this, Sic1 is able to bind to, and strongly inhibit the kinase activity of, the Cdk2-cyclin A complex. In addition, comparison of the different inhibitory patterns obtained using histone H1 or GST (glutathione S-transferase)-pRb (retinoblastoma protein) fusion protein as substrate (the latter of which recognizes both the docking site and the catalytic site of Cdk2-cyclin A) offers interesting suggestions for the inhibitory mechanism of Sic1. Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae, like overexpression of the related SIC1 gene, rescues the cell cycle-related phenotype of a sic1∆ strain. Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27Kip1 are functional homologues with a structurally conserved inhibitory domain.

Subcellular Localization of the Cyclin Dependent Kinase Inhibitor Sic1 is Modulated by the Carbon Source in Budding Yeast

“…53,54 We did not further investigate the details on how Sic1 facilitates Clb5 nuclear accumulation, however, we recall that a functional homology between Sic1 and p27 Kip1 , which on the other hand is homologous to p21 Cip1 , 55 has been recently reported from our laboratory, based on a conserved inhibitory domain between Sic1 and p27 Kip1 . 56 Experimental evidences are in favour of a two sites mechanism for binding of Sic1 to cyclin-Cdk complex: Sic1 interacts poorly with Cdk alone, but quite strongly with the cyclin-Cdk complex, making contacts both with cyclin and with the active site of the kinase, 56 with close resemblance to findings reported for the Cip/Kip proteins. 47 In conclusion this paper supports a new role of Sic1 to positively regulate cell cycle progression by facilitating cyclin-Cdk assembly and nuclear accumulation, as it has been demonstrated in mammalian cells.…”

Evolution of networks and sequences in eukaryotic cell cycle control

“…Even more distantly related homologues, such as the KRP homologues of animal Kip-Cip Cdk inhibitors, show quite clear sequence similarity over key regions known from structural biology to be central to cyclin-Cdk inhibition by p27 and known from detailed mapping studies to be critical for Cdk inhibition by KRP [95][96][97]. Figure 4 shows sequence alignments of this critical region of KRP from maize [95] and moss with animal p27, along with a presumably best-case alignment of budding yeast Sic1 and the homologous Rum1 from fission yeast [99] to the same region, based on structural considerations and modelling [52]. The alignment between the plant and animal protein is obvious and almost sure to indicate monophyly; while Sic1 and Rum1 align with each other, they do not obviously align to the p27/KRP family any better than would be expected by chance (note that even the few possibly conserved residues between Sic1 and KRPs are not generally conserved in Rum1).…”

“…A sequence alignment of animal p27 from human (Homo sapiens) and C. elegans (nematode) [98], plant KRP1 [95] from Zea mays (maize) and P. patens (moss), along with a best-case alignment of Rum1 from fission yeast (Schizosaccharomyces pombe) and Sic1 from budding yeast (Saccharomyces cerevisiae) to the same region based on structural considerations and modelling [52,98,99]. The homology between the plant KRP1 and animal p27 is likely indicative of monophyly; it is not clear that the Sic1/Rum1 alignment to KRP1/p27 is any better than would be expected by chance.…”

Identification of Clb2 Residues Required for Swe1 Regulation of Clb2-Cdc28 in Saccharomyces cerevisiae

“…The HP region also may mediate CDK inhibition by Sic1, which is functionally and structurally related to mammalian p27 (Barberis et al 2005); p27 binds the HP of cyclin A, and when expressed in S. cerevisiae can bind and inhibit Clb5-Cdc28 (Cross and Jacobson 2000). Sic1 overexpression prevents Swe1 phosphorylation of Clb5-associated Cdc28-Y19, suggesting that Sic1 and Swe1 bind overlapping sites on Clb5-Cdc28 (Keaton et al 2007).…”