Trihydrophobin 1 Interacts with PAK1 and Regulates ERK/MAPK Activation and Cell Migration

Abstract

The Rac1/Cdc42 effector, p21-activated kinase (PAK), is activated by various signaling cascades, including receptor-tyrosine kinases and integrins, and regulates a number of processes such as cell proliferation and motility. PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade, possibly because of PAK co-activation of RAF and MEK. Here we have shown that trihydrophobin 1 (TH1), originally identified as a negative regulator of A-RAF kinase, also interacted with PAK1 in cultured cells. Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus, which is consistent with our previous results. GST pulldown and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1, and the ability of the binding was enhanced along with activation of PAK1. The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction. Interestingly, TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity. Furthermore, we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells, where TH1 reduced cell migration through affecting actin and adhesion dynamics. Based on these observations, we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream region of the MAPK pathway, thereby influencing cell migration.

JWA regulates melanoma metastasis by integrin αVβ3 signaling

“…We then conducted western blot analysis to measure the protein levels of integrin a V and b 3 subunits, and the focal adhesion kinase (FAK) and its phosphorylation status, a critical mediator of integrin signaling (Mitra and Schlaepfer, 2006; van Nimwegen and van de Water, 2007). We also detected the expression of p21-activated kinase (PAK)-1 and its phosphorylation status, a downstream molecule of integrin a V b 3 signaling pathway (Zhou and Kramer, 2005;Cheng et al, 2009). Our results showed that the knockdown of JWA upregulated FAK and PAK-1 expression and activated their phosphorylation status in both B16 cells (Figure 3b, left panel) and A375 cells (Figure 3b, right panel).…”

“…The phosphorylation of FAK on Tyr397 site results in the activation of Rac. Rac then leads to the activation of PAK, which enhances actin polymerization (Zhou and Kramer, 2005;Cheng et al, 2009). Analysis of the integrin a V b 3 promoter revealed many Sp1 binding sites that are important for integrin a V b 3 transcription in B16 and A375 cells.…”

Regulation of endothelial MAPK/ERK signalling and capillary morphogenesis by low-amplitude electric field

“…The total and phosphorylated levels of ERK, MEK, p38 and JNK proteins were quantified using appropriate sandwich ELISA kits (Cell Signaling Technology, MA, USA). MEK-cRaf complex levels and free (unbound) MEK levels were quantified according to previously described protocols with modifications (see the electronic supplementary material, figure S1) [46][47][48]. The total MEK levels are presented in optical densities and phosphorylated MEK, MEK-cRaf and pMEK-cRaf levels are normalized to total MEK levels.…”

Thr-370 Is Responsible for CDK11p58 Autophosphorylation, Dimerization, and Kinase Activity

“…As a cyclin-dependent kinase, CDK11 p58 interacts with cyclin D3 to regulate G 2 /M phase cell cycle progression (14) and represses AR-mediated transactivation through phosphorylating AR to inhibit androgen-dependent proliferation of prostate cancer cells (15). CDK11 p58 is also involved in the negative regulation of estrogen receptor and vitamin D3 receptor, the other two nuclear transcription factors (16,17).…”