Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through cofilin phosphorylation downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively, activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an effector protein kinase for LIMKs downstream of Cdc42 remains to be defined. We now report evidence that LIMK1 and LIMK2 activities toward cofilin phosphorylation are stimulated in cells by the co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha-induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha phosphorylates and activates LIM kinases downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through the phosphorylation and inactivation of ADF/cofilin.     

Cloning and chromosomal localization of human Cdc42-binding protein kinase beta

The p21 GTPases, Rho and Cdc42, regulate numerous cellular functions by binding to members of a serine/threonine protein kinase subfamily. These functions include the remodeling of the cell cytoskeleton that is a feature of cell growth and differentiation. Two of these p21 GTPase-regulated kinases, the myotonic dystrophy protein kinase-related Cdc42-binding kinases (MRCKalpha and beta), have been recently characterized in rat. Both of these proteins phosphorylate nonmuscle myosin light chain, a prerequisite for the activation of actin-myosin contractility. Here we report the cDNA cloning of the human homologue of MRCKbeta, CDC42BPB, which was found by Northern blot analysis to be expressed in a wide range of tissues. The human CDC42BPB gene maps to cytogenetic band 14q32.3 by FISH analysis.     

Insensitivity to transforming growth factor-beta results from promoter methylation of cognate receptors in human prostate cancer cells (LNCaP)

Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TbetaRI), and type II (TbetaRII) TGF-beta receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TbetaRI promoter and of Sp1 in the TbetaRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.