Influence of Ras and Rho proteins on G1/S transition

Harvey rat sarcoma viral oncogene homolog (H-Ras) activates G1/S transition and cell proliferation mainly by v-raf-1 murine leukemia viral oncogene homolog 1 (c-Raf-1)/Mitogen-activated protein kinase kinases 1/2 (MEK1/2)/ Mitogen-activated protein kinases 3/1 (ERK1/2) cascade [1], [2]. ERK1 and ERK2 inhibit transducer of ERBB2, 1 (Tob1) (a suppressor of Cyclin D1 expression) by phosphorylation at Ser 152, Ser 154, and Ser 164, thus activating expression of Cyclin D1 and Mdm2 p53 binding protein homolog (MDM2) and G1/S progression [3].

Cyclin D1 forms active complexes with cyclin-dependent kinases CDK4 or CDK6. These complexes phosphorylate retinoblastoma (Rb) and inhibit its ability to suppress E2F1 transcription-factor. E2F1 induces the transcription of another Cyclins required for S-phase DNA synthesis, such as Cyclin E and Cyclin A2 [4], [5], [6].

Cyclin E forms a complex with CDK2 that also inhibits Rb and cyclin-dependent kinase inhibitor 1B (p27Kip1)by phosphorylation [5], [7].

Another pathway of H-Ras induction of Cyclin D1 expression is by activation of Phosphoinositide-3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologs(AKT(PKB)) [4].AKT(PKB) probably via activation of Conserved helix-loop-helix ubiquitous kinase (IKK-alpha) followed by phosphorylation of Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA) induces v-rel reticuloendotheliosis viral oncogene homolog A (RelA(p65 NF-kB)). NF-kB p50/p65 complex is required for Cyclin D1 expression [8], [9].

Phosphorylation of CyclinD1 by Glycogen synthase kinase 3 beta (GSK3beta) results in the ubiquitylation and degradation of Cyclin D1. However, H-Ras by PI3K/ AKT(PKB) inhibits Glycogen synthase kinase 3 beta (GSK3beta) activity, thereby enhancing Cyclin-D1 protein stability [10].

Moreover H-Ras activates Ciclin D1 expression and cell proliferation by Ral guanine nucleotide dissociation stimulator-like 2 (RGL2)/ v-ral simian leukemia viral oncogene homolog A (RalA) pathway [4], [11].

In addition, H-Ras via Raf/ MEK/ ERK and PI3K/ AKT(PKB)/ GSK3beta pathways enhances the accumulation of v-myc myelocytomatosis viral oncogene homolog (c-Myc) activity that promotes G1-phase of the cell cycle[2].

Rho family GTPases Ras homolog gene family, member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1), and Cell division cycle 42 (CDC42) are each required for progression from G1 to S phase [12].

Rac1 and CDC42 activate p21 protein (Cdc42/Rac)-activated kinase 1(Pak1). Pak1 via IKKs-independent pathway activates NF-kB p50/p65 that promotes expression of Cyclin D1 [9], [13], [14]. Possibly, Rac1 and CDC42 via Pak1 cooperate with c-Raf-1 to activate MEK1/ERK1/2 pathway and promote Cyclin D1 expression and G1 progression. Pak1 phosphorylates MEK1 on Ser298, a site important for binding of c-Raf-1 to MEK1 [15], [16], [17].

Another possible pathway of Rac1 in G1 progression is Mitogen-activated protein kinase kinase kinase 11 (MLK3(MAP3K11))/ Mitogen-activated protein kinase kinase 4 (MEK4(MAP2K4))/ Mitogen-activated protein kinase 8 (JNK1(MAPK8)) signaling [12], [18]. JNK1(MAPK8) probably by activation of Activating transcription factor 2 (ATF-2) induces Cyclin D1 expression [9].

CDC42 through ribosomal protein S6 kinase, 70kDa, polypeptide 1 (p70S6 kinase1) induces Cyclin E expression and promotes G1 progression [19].

RhoA influence on G1/S transition by two distinct mechanisms.

Firstly, geranylgeranylation of RhoA by protein geranylgeranyltransferase type I (GGTase1) is essential for repression of Cyclin-dependent kinase inhibitor 1A (p21) and p27Kip1 [20], [21], [22]. RhoA via Diaphanous homolog 1 (DIA1) increases expression of mRNA and protein levels S-phase kinase-associated protein 2 (Skp2). Skp2 promotes ubiquitin-mediated proteasomal degradation of p27Kip1 and restores G1 progression [23]. Skp2 expression is upregulated by E2F1 via positive feedback loop [24]. Rac1 via actin polymerization also increases Skp2 levels and thus controls vascular smooth muscle cell proliferation [25]. In addition, RhoA inhibits p21 expression [21], [22], [26].

Secondly, RhoA regulates expression of Cyclin D1 and Cyclin E [22], [27], [28]. RhoA activates Rho-associated, coiled-coil containing protein kinase 2 (ROCK2). ROCK2 via phosphorylation of LIM domain kinases 2 (LIMK2) and inactivation the Myosin light chain phosphatase (MLCP), enhances phosphorylation of Myosin, light chain, regulatory (MRLC) by Myosin light chain kinase (MLCK). This leads to stress fiber formation [29], [30], consequent clustering of alpha-5/beta-1 integrin and Focal adhesion kinase (FAK1) phosphorylation, followed by activation of H-Ras/ c-Raf-1/MEK1(MAP2K1), MEK2(MAP2K2)/ ERK1/2 pathway, leading to the elevation of Cyclin D1 [31]. Also phosphorylation of LIMK2 increases Cyclin A2 expression [30].

In addition, RhoA via ROCK2 and DIA1 activates signal transducer and activator of transcription 3 (STAT3) by phosphorylation of Tyr-705 and Ser-727 that accompanied STAT3 nuclear translocation. STAT3 induces NF-kB and Cyclin D1 transcription and is involved in NF-kB nuclear translocation [32].

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