Cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), is targeted for proteasomal degradation by phosphorylation-dependent multiubiquitylation via the ubiquitin ligase SCF(hCdc4). SCF ubiquitin ligases are composed of a core of conserved subunits and one variable subunit (an F box protein) involved in substrate recognition. We show here that multiubiquitylation of cyclin E requires the sequential function of two distinct splice variant isoforms of the F box protein hCdc4 known as alpha and gamma. SCF(hCdc4alpha) binds a complex containing cyclin E, Cdk2, and the prolyl cis/trans isomerase Pin1 and promotes the activity of Pin1 without directly ubiquitylating cyclin E. However, due to the action of this SCF(hCdc4alpha)-Pin1 complex, cyclin E becomes an efficient ubiquitylation substrate of SCF(hCdc4gamma). Furthermore, in the context of Cdc4alpha and cyclin E, mutational data suggest that Pin1 isomerizes a noncanonical proline-proline bond, with the possibility that Cdc4alpha may serve as a cofactor for altering the specificity of Pin1.

Although scaffolding is a major regulator of mitogen-activated protein kinase (MAPK) pathways, scaffolding proteins are poorly understood. During yeast mating, MAPK Fus3p is phosphorylated by MAPKK Ste7p, which is activated by MAPKKK Ste11p. This MAPK module interacts with the scaffold molecule Ste5p. Here we show that Ste11p and Ste7p were predominantly cytoplasmic proteins, while Ste5p and Fus3p were found in the nucleus and the cytoplasm. Ste5p, Ste7p and Fus3p also localized to tips of mating projections in pheromone-treated cells. Using fluorescence recovery after photobleaching (FRAP), we demonstrate that Fus3p rapidly shuttles between the nucleus and the cytoplasm independently of pheromones, Fus3p phosphorylation and Ste5p. Membrane-bound Ste5p can specifically recruit Fus3p and Ste7p to the cell cortex. Ste5p remains stably bound at the plasma membrane, unlike activated Fus3p, which dissociates from Ste5p and translocates to the nucleus.

Our results suggest that, in response to multiple extracellular signals, phosphorylation of Ste11p by Ste20p removes an amino-terminal inhibitory domain, leading to activation of the Ste11 protein kinase. This mechanism may serve as a paradigm for the activation of mammalian MEKKs.

Site-specific activation of the Rho-type GTPase Cdc42p is critical for the establishment of cell polarity. Here we investigated the role and regulation of the GTPase-activating enzymes (GAPs) Bem2p and Bem3p for Cdc42p activation and actin polarization at bud emergence in Saccharomyces cerevisiae. Bem2p and Bem3p are localized throughout the cytoplasm and the cell cortex in unbudded G1 cells, but accumulate at sites of polarization after bud emergence. Inactivation of Bem2p results in hyperactivation of Cdc42p and polarization toward multiple sites. Bem2p and Bem3p are hyperphosphorylated at bud emergence most likely by the Cdc28p-Cln2p kinase. This phosphorylation appears to inhibit their GAP activity in vivo, as non-phosphorylatable Bem3p mutants are hyperactive and interfere with Cdc42p activation. Taken together, our results indicate that Bem2p and Bem3p may function as global inhibitors of Cdc42p activation during G1, and their inactivation by the Cdc28p/Cln kinase contributes to sitespecific activation of Cdc42p at bud emergence.

Scaffold proteins play a major role in regulating MAP kinase pathways. In yeast, the Mpk1p-MAP kinase pathway functions to maintain the integrity of the cytoskeleton and the cell wall. In this module, the MEKK Bck1p functions upstream of the MEKs Mkk1p and Mkk2p, which in turn activate the MAP kinase Mpk1p. Mpk1p regulates several nuclear targets, including the transcription factors Rlm1p and SBF, and the two HMG1-like proteins NHP6A and NHP6B. Here we show that Mpk1p constitutively shuttles between the nucleus and the cytoplasm, and both Mpk1p and Mkk1p localize to sites of polarized growth in a Spa2p-dependent manner. Spa2p belongs to a group of proteins that includes Bni1p, Bud6p, and Pea2p, which are involved in the dynamic organization of the actin cytoskeleton during polarized growth. FRAP analysis shows that Spa2p-GFP is stably anchored at bud tips, whereas Mpk1p binds transiently. Spa2p interacts with Mkk1p and Mpk1p, and membrane bound Spa2p is sufficient to recruit Mkk1p and Mpk1p but not other MAP kinases to the cell cortex. Taken together, these results suggest that Spa2p functions as a scaffold-like protein for the cell wall integrity pathway during polarized growth.