Rac1/p21‐activated kinase pathway controls retinoblastoma protein phosphorylation and E2F transcription factor activation in B lymphocytes

Abstract

Small GTPases of the Ras superfamily are capable of activating E2F-dependent transcription leading to cell proliferation, but the molecular mechanisms are poorly understood. In this study, using immortalized chicken DT40 B cell lines to investigate the role of the Vav/Rac signalling cascade on B cell proliferation, it is shown that the proliferative response triggered by B cell receptor activation is dramatically reduced in the absence of Vav3 expression. Analysis of this proliferative defect shows that in the absence of Vav3 expression, retinoblastoma protein (RB) phosphorylation and the subsequent E2F activation do not take place. By combining pharmacological and genetic approaches, phosphatidylinositol-3-kinase and phospholipase Cc2 (PLCc2) were identified as the key regulatory signalling molecules upstream of the Vav3/Rac pathway leading to RB phosphorylation and E2F transcription factor activation. Additionally, vav3À/À and plcc2 À/À DT40 B cells were not able to activate the RB-E2F complex wild-type phenotype when these genetically modified cells were transfected with constitutively active forms of RhoA or Cdc42. However, when these knockout cells were transfected with different constitutively active versions of PLCc, Vav or Rac1, not only activation of the RB-E2F complex wild-type phenotype was recovered but also the cellular proliferation. Furthermore, by evaluating the effect of two known effector mutants of Rac1 (Rac1 Q61L/F37A and Rac1), the RB-E2F complex activation dependency on p21-activated kinase (PAK) and protein kinase Ce (PKCe) activities was established, being independent of both actin cytoskeleton reorganization and Ras activity. These results suggest that PAK1 and PKCe may be potential therapeutic targets to stop uncontrolled B cell proliferation mediated by the Vav/Rac pathway.Abbreviations BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N 0 ,N 0 -tetraacetic acid tetrakis(acetoxymethyl ester); BCR, B cell receptor; CDK, cyclindependent kinase; DAG, diacylglycerol; GEF, guanine nucleotide exchange factor; PAK, p21-activated kinase; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PKD, protein kinase D; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; RasGRP, RAS guanyl releasing protein; RB, retinoblastoma protein.

Identification of Biological Targets of Therapeutic Intervention for Hepatocellular Carcinoma by Integrated Bioinformatical Analysis

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