Faculty Bibliography

Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment

The cyclosome/anaphase-promoting complex is a multisubunit ubiquitin ligase that targets for degradation mitotic cyclins and some other cell cycle regulators in exit from mitosis. It becomes enzymatically active at the end of mitosis. The activation of the cyclosome is initiated by its phosphorylation, a process necessary for its conversion to an active form by the ancillary protein Cdc20/Fizzy. Previous reports have implicated either cyclin-dependent kinase 1-cyclin B or polo-like kinase as the major protein kinase that directly phosphorylates and activates the cyclosome. These conflicting results could be due to the use of partially purified cyclosome preparations or of immunoprecipitated cyclosome, whose interactions with protein kinases or ancillary factors may be hampered by binding to immobilized antibody. To examine this problem, we have purified cyclosome from HeLa cells by a combination of affinity chromatography and ion exchange procedures. With the use of purified preparations, we found that both cyclin-dependent kinase 1-cyclin B and polo-like kinase directly phosphorylated the cyclosome, but the pattern of the phosphorylation of the different cyclosome subunits by the two protein kinases was not similar. Each protein kinase could restore only partially the cyclin-ubiquitin ligase activity of dephosphorylated cyclosome. However, following phosphorylation by both protein kinases, an additive and nearly complete restoration of cyclin-ubiquitin ligase activity was observed. It is suggested that this joint activation may be due to the complementary phosphorylation of different cyclosome subunits by the two protein kinases

BACKGROUND: Previous studies have shown that low levels of p27(Kip1), an inhibitor of G1 cyclin-dependent kinases, are associated with high aggressiveness and poor prognosis in a variety of cancers. Decreased levels of p27 are caused, at least in part, by acceleration of the rate of its ubiquitin-mediated degradation. In cultured cells and cell-free biochemical systems, it has been shown that p27 is targeted for degradation by a ubiquitin ligase complex that contains Skp2 (S-phase kinase-associated protein 2) as the specific substrate-recognizing and rate-limiting subunit. This investigation was undertaken to examine the possible relation between levels of p27 and of its specific ubiquitin ligase subunit Skp2 in human cancers. METHODS: Quick-frozen colorectal tumor samples from 20 patients were homogenized at 0 degrees C in buffer containing a mixture of protease inhibitors. Samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, transferred to nitrocellulose, and probed with highly specific monoclonal antibodies directed against Skp2 and p27. The expression of Skp2 also was examined by immunohistochemistry using formalin fixed, paraffin embedded tissue sections from the same cases. RESULTS: A strongly significant inverse correlation was found between levels of Skp2 and p27 (r = -0.812; P < 0.0001). Thus, decreased levels of p27 were associated with strongly increased levels of Skp2, whereas high levels of p27 coincided with low levels of Skp2. Immunohistochemical examination of Skp2 expression agreed with immunoblot analysis in 89% of cases. CONCLUSIONS: The results are compatible with the notion that increased expression of Skp2 may have a causative role in decreasing the levels of p27 in aggressive colorectal carcinomas.

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded in late G1 phase by the ubiquitin pathway, allowing CDK activity to drive cells into S phase. Ubiquitinylation of p27 requires its phosphorylation at Thr 187 (refs 3, 4) and subsequent recognition by S-phase kinase associated protein 2 (Skp2; refs 5-8), a member of the F-box family of proteins that associates with Skp1, Cul-1 and ROC1/Rbx1 to form an SCF ubiquitin ligase complex. However, in vitro ligation of p27 to ubiquitin could not be reconstituted by known purified components of the SCFSkp2 complex. Here we show that the missing factor is CDK subunit 1 (Cks1), which belongs to the highly conserved Suc1/Cks family of proteins that bind to some CDKs and phosphorylated proteins and are essential for cell-cycle progression. Human Cks1, but not other members of the family, reconstitutes ubiquitin ligation of p27 in a completely purified system, binds to Skp2 and greatly increases binding of T187-phosphorylated p27 to Skp2. Our results represent the first evidence that an SCF complex requires an accessory protein for activity as well as for binding to its phosphorylated substrate

Degradation of the mammalian cyclin-dependent kinase (CDK) inhibitor p27 is required for the cellular transition from quiescence to the proliferative state. The ubiquitination and subsequent degradation of p27 depend on its phosphorylation by cyclin-CDK complexes. However, the ubiquitin-protein ligase necessary for p27 ubiquitination has not been identified. Here we show that the F-box protein SKP2 specifically recognizes p27 in a phosphorylation-dependent manner that is characteristic of an F-box-protein-substrate interaction. Furthermore, both in vivo and in vitro, SKP2 is a rate-limiting component of the machinery that ubiquitinates and degrades phosphorylated p27. Thus, p27 degradation is subject to dual control by the accumulation of both SKP2 and cyclins following mitogenic stimulation

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor