Assembly of Scaffold-mediated Complexes Containing Cdc42p, the Exchange Factor Cdc24p, and the Effector Cla4p Required for Cell Cycle-regulated Phosphorylation of Cdc24p

Abstract

In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclindependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTPCdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle.

Yeast and fungal morphogenesis from an evolutionary perspective

“…However, Bem1 appears to share several domains with proteins in animals that are directly regulated by Rho family GTPases, such as p47Phox [71] and the conserved polarity protein Par6 [72]. Precisely how Bem1 functions in cell polarization remains an open question, but existing data suggest that Bem1 interacts with Cdc42 GTP , the Cdc42 GEF Cdc24 and the Cdc42 effectors Cla4 and Ste20 [73,74] and may mediate a feedback loop linking Cdc42 activation and GEF activation. During bud formation, the two polarization mechanisms described above are partially redundant, but their coupling brings both temporal precision and stability to polarity establishment [69,75].…”

“…Ste20 and Cla4 have been shown to phosphorylate and activate type I myosins Myo3 and Myo5 [98], which in turn have a role in the internalization step of endocytosis via actin patches [99,100]. Cla4 is also the major kinase that phosphorylates the GEF Cdc24, although the exact role for this phosphorylation is still controversial [74,101]. Cla4 plays a critical role in the assembly of the septin ring at the bud neck [102].…”

All Phox Homology (PX) Domains from Saccharomyces cerevisiae Specifically Recognize Phosphatidylinositol 3-Phosphate

“…Alternatively, homo-and/or hetero-oligomerization of proteins containing low affinity PX domains could enhance their effective PtdIns-3-P binding affinity or avidity. For example, Bem1p is a scaffolding protein that forms complexes with several proteins, some of which have PH domains (24) at sites of bud emergence. Perhaps the clearest illustration of this point, however, is seen with the sorting nexins that regulate membrane traffic.…”

The Cdc42 Effectors Ste20, Cla4, and Skm1 Down-Regulate the Expression of Genes Involved in Sterol Uptake by a Mitogen-activated Protein Kinase-independent Pathway

“…PAKs carry a conserved Cdc42/ Rac-interactive binding (CRIB) domain that mediates binding to Cdc42 and regulates their activity (Cvrckova et al, 1995;Peter et al, 1996;Leberer et al, 1997;Lamson et al, 2002;Ash et al, 2003). Ste20 and Cla4 also bind to Bem1, a scaffold protein that brings Cdc42, its activator Cdc24 and either Ste20 or Cla4, into proximity (Leeuw et al, 1998;Gulli et al, 2000;Bose et al, 2001;Yamaguchi et al, 2007). In addition, phosphoinositide-binding domains promote the association with membrane lipids.…”