Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro.

D-type cyclin-dependent kinases (Cdk4 and Cdk6) regulate the G1 to S phase progression of the mammalian cell cycle. It has been suggested that Cdk4 and Cdk6 may have distinct functions in vivo, even though they are indistinguishable biochemically. Here we show that although these Cdks phosphorylate multiple residues in pRB, they do so with different residue selectivities in vitro; Thr821 and Thr826 are preferentially phosphorylated by Cdk6 and Cdk4, respectively. This raises the possibility different substrate specificities lead to their different roles in the regulation of cellular events. Furthermore, our results indicate the new concept that Cdk itself contributes to substrate recognition.

Cdk-mediated phosphorylation of pRB regulates HDAC binding in vitro.

Retinoblastoma protein (pRB) controls the G1/S transition in the cell cycle by binding and inactivating E2F transcription factor. pRB changes the chromatin structure at the E2F-responsive promoter by recruiting histone deacetylase (HDAC) to the pRB-E2F complex, thus controlling the transcriptional activity of E2F. Cyclin-dependent kinases (Cdks) phosphorylate pRB and disrupt association between pRB and E2F. We investigated the effects of pRB phosphorylation on HDAC-1 binding in vitro. Phosphorylation of pRB by Cdk4-cyclin D2, Cdk2-cyclin E, and Cdk2-cyclin A inhibited association of pRB with HDAC. Among these Cdks, Cdk4-cyclin D2 showed particularly effective inhibition of pRB-HDAC complex formation. Using pRB mutants with various deletions in the N- and C-terminal domains, we found that both the pocket and C-terminal domains are important for regulating association between pRB and HDAC.

The K252a derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth of RAS transformants.

BACKGROUND: Oncogenic RAS mutants such as v-Ha-RAS activate members of Rac/CDC42-dependent kinases (PAKs) and appear to contribute to the development of more than 30% of all human cancers. PAK1 activation is essential for oncogenic RAS transformation, and several chemical compounds that inhibit Tyr kinases essential for the RAS-induced activation of PAK1 strongly suppress RAS transformation either in cell culture or in vivo (nude mice). Although we have developed a cell-permeable PAK-specific peptide inhibitor called WR-PA18, so far no chemical (metabolically stable) compound has been developed that directly inhibits PAK1 in a highly selective manner. Thus, we have explored such a PAK1 inhibitor(s) among synthetic derivatives of an adenosine triphosphate antagonist. RESULTS: From the naturally occurring adenosine triphosphate antagonist K252a, we have developed two bulky derivatives, called CEP-1347 and KT D606 (a K252a dimer), which selectively inhibit PAKs or mixed-lineage kinases both in vitro and in cell culture and convert v-Ha-RAS-transformed NIH 3T3 cells to flat fibroblasts similar to the parental normal cells. Furthermore, these two K252a analogues suppress the proliferation of v-Ha-RAS transformants, but not the normal cells. CONCLUSION: These bulky adenosine triphosphate antagonists derived from K252a or related indolocarbazole compounds such as staurosporine would be potentially useful for the treatment of RAS/ PAK1-induced cancers, once their anti-PAK1 activity is significantly potentiated by a few additional chemical modifications at the sugar ring suggested in this paper.