In S. cerevisiae, the G1/S transition requires Cdc4p, Cdc34p, Cdc53p, Skp1p, and the Cln/Cdc28p cyclin-dependent kinase (Cdk). These proteins are thought to promote the proteolytic inactivation of the S-phase Cdk inhibitor Sic1p. We show here that Cdc4p, Cdc53p, and Skp1p assemble into a ubiquitin ligase complex named SCFCdc4p. When mixed together, SCFCdc4p subunits, E1 enzyme, the E2 enzyme Cdc34p, and ubiquitin are sufficient to reconstitute ubiquitination of Cdk-phosphorylated Sic1p. Phosphorylated Sic1p substrate is specifically targeted for ubiquitination by binding to a Cdc4p/Skp1p subcomplex. Taken together, these data illuminate the molecular basis for the G1/S transition in budding yeast and suggest a general mechanism for phosphorylation-targeted ubiquitination in eukaryotes.

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of beta-catenin (reviewed in refs 1-3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore-microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.

We have explored the role of the tyrosine kinase c-Src in cellular adhesion. Fibroblasts derived from src-~-mice (src-/-fibroblasts) exhibit a reduced rate of spreading on fibronectin. This defect is rescued by expression of wild-type chicken c-Src. Analyses of mutants suggest that c-Src increases the rate of cell spreading in src-/-fibroblasts through a kinase-independent mechanism requiring both the SH3 and SH2 domains. To further address the role of c-Src in adhesion, we examined the activity and subcellular distribution of c-Src during the adhesion of fibroblasts on fibronectin. We observed a transient increase in the specific kinase activity of c-Src accompanied by the partial dephosphorylation of the negative regulatory site Y527. Activation of c-Src is followed by its redistribution to newly formed focal adhesions. These results suggest that the enzymatic activity and subcellular distribution of c-Src are coordinately regulated during cellular adhesion and that c-Src can affect adhesion by a kinase-independent mechanism.

We have characterized the mechanism by which the subcellular distribution of c‐Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c‐Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c‐Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c‐Src inside the cell. The first 251 amino acids of c‐Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c‐Src in focal adhesions resides in the amino‐terminal half. Point mutations and deletions in the first 251 amino acids of c‐Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino‐terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non‐catalytic portion of c‐Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c‐Src may regulate focal adhesions and cellular adhesion by a kinase‐independent mechanism.