Relationship of p21‐activated kinase (PAK) and filopodia to persistence and oncogenic transformation

Abstract

Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

PAK–PIX interactions regulate adhesion dynamics and membrane protrusion to control neurite outgrowth

“…2H), possibly by displacing active PAK away from point contacts to filopodial tips. This is consistent with previous studies implicating PAK signaling in the regulation of both axonal and dendritic filopodial extension (Heckman et al, 2009;Kayser et al, 2008;Robles et al, 2005). These results suggest that PAK2 regulates actin filament polymerization, but the relevant targets of PAK at filopodial tips remain unknown.…”

3,3′,4,4′,5,5′-Hexahydroxystilbene Impairs Melanoma Progression in a Metastatic Mouse Model

“…In addition, a key regulator of cell migration, paxillin, was as well found to be regulated in M24met and 1205Lu melanoma cell lines (Table 2). P21, the known activator of PAK, may indirectly affect cytoskeletal organization and cell migration through regulation of the Rho pathway (Heckman et al, 2009) and is involved in cell-cycle progression. Therefore p21, verified to be affected by M8 with western blotting and shotgun proteomics, provides an important interference partner for the compound M8.…”

CD48 on hematopoietic progenitors regulates stem cells and suppresses tumor formation

“…Hyperactivation of PAK is known to be involved in the formation of several cancer types, [35][36][37][38] and the increased activation of Pak1 in the hematopoietic progenitors from CD48 Ϫ/Ϫ mice led us to speculate increased Pak1 activation may also play a role in the lymphoma development in the CD48-null mice. Splenocytes from WT mice and CD48 Ϫ/Ϫ mice with lymphoma in the spleen were isolated and stained with either CD19 or early B-cell lineage Ag, and the phosphorylation status of Pak1 and Erk was examined ( Figure 5D-E).…”