Cla4p, a Saccharomyces cerevisiae Cdc42p-Activated Kinase Involved in Cytokinesis, Is Activated at Mitosis†

Abstract

Yeasts have three functionally redundant G 1 cyclins required for cell cycle progression through G 1 . Mutations in GIN4 and CLA4 were isolated in a screen for mutants that are inviable with deletions in the G 1 cyclins CLN1 and CLN2. cln1 cln2 cla4 and cln1 cln2 gin4 cells arrest with a cytokinesis defect; this defect was efficiently rescued by CLN1 or CLN2 expression. GIN4 encodes a protein with strong homology to the Snf1p serine/ threonine kinase. Cla4p is homologous to mammalian p21-activated kinases (PAKs) (kinases activated by the rho-class GTPase Rac or Cdc42). We developed a kinase assay for Cla4p. Cla4p kinase was activated in vivo by the GTP-bound form of Cdc42p. The specific activity of Cla4p was cell cycle regulated, peaking near mitosis. Deletion of the Cla4p pleckstrin domain diminished kinase activity nearly threefold and eliminated in vivo activity. Deletion of the Cla4p Cdc42-binding domain increased kinase activity nearly threefold, but the mutant only weakly rescued cla4 function in vivo. This suggests that kinase activity alone is not sufficient for full function in vivo. Deletion of the Cdc42-binding domain also altered the cell cycle regulation of kinase activity. Instead of peaking at mitosis, the mutant kinase activity exhibited reduced cell cycle regulation and peaked at the G 1 /S border. Cla4p kinase activity was not reduced by mutational inactivation of gin4, suggesting that Gin4p may be downstream or parallel to Cla4p in the regulation of cytokinesis.

The p21-activated Protein Kinase-related Kinase Cla4 Is a Coincidence Detector of Signaling by Cdc42 and Phosphatidylinositol 4-Phosphate

“…6A), as reported previously (46). These loss of function phenotypes were not due to complete loss of Cla4 kinase activity, because the PH⌬ mutant has been shown previously to have detectable (albeit reduced) kinase activity, whereas the PBD⌬ mutant has elevated kinase activity relative to wild type Cla4 (46).…”

“…In contrast, expression of mutant Cla4-GFP in which the PH domain or PBD was deleted failed to rescue the elongated bud phenotype (only 40% of cells had normal buds, similar to empty vector controls; Fig. 6A), as reported previously (46). These loss of function phenotypes were not due to complete loss of Cla4 kinase activity, because the PH⌬ mutant has been shown previously to have detectable (albeit reduced) kinase activity, whereas the PBD⌬ mutant has elevated kinase activity relative to wild type Cla4 (46).…”

“…The rationale for doing so was severalfold. In contrast to other proteins such as Boi1 that bind Cdc42 by poorly understood mechanisms, Cla4 uses its conserved PBD to bind Cdc42 (46). Furthermore, Cla4 probably binds Cdc42 by a mechanism highly similar to that used by mammalian PAK1 because the residues in PAK1 that bind Cdc42 are conserved in Cla4 (Fig.…”

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

“…In S. cerevisiae, there are five Cdc42 effectors with recognizable CRIB domains ( Figure 7A). Interestingly, Cla4 and Skm1 have PH domains at a position similar to that of the Ste20 BR domain, and the PH domain of Cla4 is essential for its function (Benton et al, 1997;Wild et al, 2004). Therefore, we wondered if the other two Cdc42 targets, Gic1 and Gic2, might also contain membrane-binding domains in an analogous position.…”

Yeast polo-like kinases: functionally conserved multitask mitotic regulators

“…A PAK homolog Cla4 (Benton et al, 1997) directly phosphorylates and regulates Swe1 stability . In addition, Clb-Cdc28 complexes associate with and phosphorylate Swe1 (Sia et al, 1998;McMillan et al, 2002), suggesting the existence of a positive feedback mechanism.…”