RACK1 regulates G1/S progression by suppressing Src kinase activity

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.     

E2F mediates sustained G2 arrest and down-regulation of Stathmin and AIM-1 expression in response to genotoxic stress

Exposure of cells to genotoxic agents results in activation of checkpoint pathways leading to cell cycle arrest. These arrest pathways allow repair of damaged DNA before its replication and segregation, thus preventing accumulation of mutations. The tumor suppressor retinoblastoma (RB) is required for the G(1)/S checkpoint function. In addition, regulation of the G(2) checkpoint by the tumor suppressor p53 is RB-dependent. However, the molecular mechanism underlying the involvement of RB and its related proteins p107 and p130 in the G(2) checkpoint is not fully understood. We show here that sustained G(2)/M arrest induced by the genotoxic agent doxorubicin is E2F-dependent and involves a decrease in expression of two mitotic regulators, Stathmin and AIM-1. Abrogation of E2F function by dominant negative E2F abolishes the doxorubicin-induced down-regulation of Stathmin and AIM-1 and leads to premature exit from G(2). Expression of the E7 papilloma virus protein, which dissociates complexes containing E2F and RB family members, also prevents the down-regulation of these mitotic genes and leads to premature exit from G(2) after genotoxic stress. Furthermore, genotoxic stress increases the levels of nuclear E2F-4 and p130 as well as their in vivo binding to the Stathmin promoter. Thus, functional complexes containing E2F and RB family members appear to be essential for repressing expression of critical mitotic regulators and maintaining the G(2)/M checkpoint.     

Distinct cellular factors regulate the c-myb promoter through its E2F element

Most E2F-driven promoters are transiently activated around the G(1)/S transition. Although the promoter for the c-myb proto-oncogene harbors an E2F element, it is induced early in G(1) following entry into the cell cycle. Furthermore, this promoter remains active throughout subsequent cell cycles. Since E2F sites function as repressor elements during G(1) (due to the association of pRb with E2F factors), we investigated whether the E2F element in the c-myb promoter is regulated differently than E2F elements in promoters that are repressed during G(1). By gel shift analysis, the E2F element from the c-myb promoter was found to form a unique complex, referred to as E2Fmyb-sp, which was not observed with E2F elements from several other promoters. Antibodies to DP-1, E2F1 to -5, p107, or pRb failed to either supershift or block E2Fmyb-sp complex formation. Methylation interference experiments indicate that the DNA contact residues for the E2Fmyb-sp complex are distinct from but overlapping with residues required for the binding of E2F proteins. In addition to the identification of E2Fmyb-sp, we have found that SP-1 binds to the c-myb E2F element. Functional studies revealed that E2Fmyb-sp and/or SP-1 are required to achieve full activation of the c-myb promoter in different cell types and to maintain elevated expression of the c-myb promoter during G(1) in NIH 3T3 cells. These studies demonstrate that E2F elements can be regulated differently through the binding of unique sets of proteins.     

Human TopBP1 participates in cyclin E/CDK2 activation and preinitiation complex assembly during G1/S transition

Human TopBP1 with eight BRCA1 C terminus domains has been mainly reported to be involved in DNA damage response pathways. Here we show that TopBP1 is also required for G(1) to S progression in a normal cell cycle. TopBP1 deficiency inhibited cells from entering S phase by up-regulating p21 and p27, resulting in down-regulation of cyclin E/CDK2. Although co-depletion of p21 and p27 with TopBP1 restored the cyclin E/CDK2 kinase activity, however, cells remained arrested at the G(1)/S boundary, showing defective chromatin-loading of replication components. Based on these results, we suggest a dual role of TopBP1 necessary for the G(1)/S transition: one for activating cyclin E/CDK2 kinase and the other for loading replication components onto chromatin to initiate DNA synthesis.     

Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G(2)/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G(2)/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G(2)/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G(2)/M but have different regulatory mechanisms and function at distinct times.