Degradation of B-Myb by ubiquitin-mediated proteolysis: involvement of the Cdc34-SCF(p45Skp2) pathway

B-Myb, a highly conserved member of the Myb oncoprotein family, is a 110 kDa sequence-specific DNA binding protein expressed in virtually all proliferating cells. B-myb expression reaches its maximum at the G1/S phase boundary and during the S phase of the cell cycle. We have previously shown that B-Myb activity is cell cycle regulated and it is controlled by the antagonistic effects of cyclin D1 and A. Here we show that ectopic expression of cyclin A causes a pronounced reduction of B-Myb protein level. We provide evidence that in addition to triggering B-Myb activity an important effect of cyclin A is to facilitate multiple ubiquitination of B-Myb. The C-terminal domain of B-Myb is of key importance in mediating this effect of cyclin A. Contrary to full-length B-Myb, a C-terminal deletion mutant displays activity irrespective of cyclin A expression, does not undergo ubiquitination, and its half-life is not affected by cyclin A. Ectopic expression of either Cdc34 or the F-box protein p45Skp2, respectively the E2 and E3 components of a ubiquitination pathway that regulates the G1/S transition, accelerates degradation of B-Myb. We show that B-Myb physically and functionally interacts with components of the Cdc34-SCFp45Skp2 ubiquitin pathway and propose that B-Myb degradation may be required for controlling the correct alternation of events during progression through the cell division cycle. Oncogene (2000).     

E1A modulates phosphorylation of p130 and p107 by differentially regulating the activity of G1/S cyclin/CDK complexes

We have previously shown that the adenoviral 12S E1A protein modulates the phosphorylation status of p130 and p107 without apparent changes in the cell cycle dependent phosphorylation of the retinoblastoma protein. Here we report on the mechanisms by which E1A modifies differentially the phosphorylation status of pocket proteins. In human U-2 OS osteosarcoma cells transiently expressing E1A, ectopic expression of D-type cyclins alone or combined, but not cyclins E and/or A, fully rescues E1A-mediated block in hyperphosphorylation of p130 to form 3. However, cyclins E and A, individually or together, induce hyperphosphorylation of p130 to species with intermediate mobility. Phosphopeptide maps indicate that E1A inhibits phosphorylation of sites phosphorylatable by CDKs. One of these sites is Ser-1044. The effects of blocking the activities of endogenous and exogenous cyclins with p16 and dominant negative CDK2 in E1A expressing cells further indicate that p130 is phosphorylated by both D-type cyclin and cyclin E/CDK complexes and that E1A modulates the activity of these G1/S CDKs by independent mechanisms. Stable expression of E1A in MC3T3-E1 cells leads to downregulation of D-type cyclins, and upregulation of cyclins E and A. This is accompanied by increased CDK2 kinase activity. Downregulation of D-type cyclins in these cells correlates with a block on both p130 hyperphosphorylation to form 3 and hyperphosphorylation of p107. This is rescued by D-type cyclins but not by cyclin E. In addition, we show that the upregulation of cyclins E and A is at least partially dependent on an intact pocket protein/E2F pathway, but downregulation of D-type cyclins is not. Moreover, we provide evidence that while the lack of a functional pRB pathway also results in a block on hyperphosphorylation of p130 to form 3, this is not sufficient to induce constitutive expression of p130 form 2b.     

Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to activation of Cdk2 complexes in late G1.

Progression of cells through the G1 phase of the cell cycle requires cyclin D:Cdk4/6 and cyclin E:Cdk2 complexes; however, the duration and ordering of these complexes remain unclear. To address this, we synthesized a peptidyl mimetic of the Cdk4/6 inhibitor, p16INK4a that contained an NH2-terminal TAT protein transduction domain. Transduction of TAT-p16 wild-type peptides into cells resulted in the loss of active, hypophosphorylated pRb and elicited an early G1 cell cycle arrest, provided cyclin E:Cdk2 complexes were inactive. We conclude that cyclin D:Cdk4/6 activity is required for early G1 phase cell cycle progression up to, but not beyond, activation of cyclin E:Cdk2 complexes at the restriction point and is thus nonredundant with cyclin E:Cdk2 in late G1.     

p107 Expression in colorectal tumours rises during carcinogenesis and falls during invasion

p107 Links to cyclin A/CDK2 (cyclin-dependent kinase 2) and cyclin E/CDK2 that are important cell cycle regulators. However, p107 expression remains unclear in almost all kinds of human solid tumours. To clarify the expression of p107 in colorectal tumours, 22 normal mucosae, 9 hyperplastic polyps, 60 adenomas, 198 primary carcinomas, 21 lymph-nodal metastases, and 10 hepatic metastases were immunohistochemically stained for p107, cyclin A, cyclin E, CDK2 and Ki67. Results were measured using labelling indices (LIs). p107 LIs surpassed the highest value in normal tissues in six of nine hyperplastic polyps, 54 of 60 adenomas, 144 of 198 primary cancers, 13 of 21 nodal foci and three of 10 hepatic foci. p107 LIs also apparently rose from normal through hyperplasia and adenoma to early carcinoma. However, they declined in liver-metastatic foci, and in primary cancers showing large size, mucinous type, venous invasion, lymphatic invasion, poorly differentiated type, deep invasion, lymph-nodal metastasis, hepatic metastasis or advanced stage. Low p107 LIs were also linked to a poor survival, particularly in stage-III patients. As the p107 LI gradually rose, the CDK2 (in primary cancers only), cyclin A, cyclin E and Ki67 LIs were elevated concurrently-in both adenomas and primary cancers. Thus, in colorectal tumours, p107 expression rises abnormally and gradually during carcinogenesis and then falls during invasion, and thereby probably perturbs the cell-cycle control and promotes carcinogenesis and invasion. Clinically, reduced p107 may indicate a poorer prognosis.     

Insufficient effect of p27(KIP1) to inhibit cyclin D1 in human esophageal cancer in vitro

The cell cycle is controlled by protein complexes composed of cyclins and cyclin-dependent kinases. p27KIP1 (p27) is one of the Kip/Cip family cyclin-dependent kinase inhibitory proteins which negatively regulate cell cycle progression, and have been proposed as candidate tumor suppressor genes. To examine the role of p27 in the development of human esophageal squamous cell carcinoma (ESCC), we performed Western blot and immunoprecipitation analyses of the levels of expression of p27 protein in a series of ESCC cell lines. This protein was expressed at various levels in these cell lines during exponential growth. p27 level was significantly associated with that of cyclin D1, but not of cyclin E. Further cell cycle synchronization studies demonstrated that p27 was free or bound with affinity to cyclin E-CDK2 more than to cyclin D1-CDK4 or cyclin D1-CDK6. It is known that overexpression of cyclin D1 rather than cyclin E is involved in the pathogenesis of ESCC. Our findings indicated that high expression of p27 throughout the G1 to S phase may inhibit more likely cyclin E, than cyclin D1, which promotes tumor growth of esophageal squamous cell carcinoma.