PAK4 and αPIX determine podosome size and number in macrophages through localized actin regulation

Abstract

Podosomes are actin-rich adhesion structures typical for monocytic cells and are implicated in migration and invasion. Major modes of podosome regulation include RhoGTPase signaling and actin regulatory pathways. However, it is not clearly understood how these signals induce highly localized changes in podosome formation and dynamics. Here, we show that the RhoGTPase effector PAK4, a member of the p21 associated kinase family, and its regulator alphaPIX (PAK-interacting exchange factor), are central to podosome formation in primary human macrophages. Immunofluorescence, biochemical and microarray data indicate that PAK4 acts as physiological regulator of podosomes in this system. Accordingly, transfection of a specific shRNA, as well as expression of PAK4 truncation mutants, resulted in reduced numbers of podosomes per cell. Moreover, expression of kinase active or inactive PAK4 mutants enhanced or reduced the size of individual podosomes, respectively, indicating a modulatory influence of PAK4 kinase activity on podosome size. Similar to the results gained with PAK4, cellular/overexpressed PIX was shown to be closely associated with podosomes. Moreover, both overexpression of alphaPIX wt and a mutant lacking the SH3 domain led to coalescence of podosomes. In sum, we propose that PAK4 and alphaPIX can induce highly localized changes in actin dynamics and thereby regulate size and number of podosomes in primary human macrophages.

Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts

“…The efficient siRNAmediated depletion of GIT1 remained without any effect on sealing-zone integrity. Similarly, the knockdown of the Rho GEF ␣ PIX or ␤ PIX, the other PIX member that was not detected as a Src target, remained without any effect, although podosomal belts appeared less compact, consistent with the previous report (22) (Fig. S2 A and B).…”

Rho GTPases in osteoclasts: Orchestrators of podosome arrangement

“…In these cells, PAK4 kinase activity regulates the size of individual podosomes suggesting that PAK4 activity may interfere with F-actin polymerization. In addition, PAK4 mutants impaired in GTPase binding diminish the number of podosome per cell (Gringel et al, 2006). Since Rho GTPases affect PAK4 localization rather than its activity (Abo et al, 1998), it suggests that Rho GTPases may act to correctly localize PAK proteins to podosome in order to modulate their dynamic and organization.…”

“…The recruitment of PIX requires binding to GIT1 and its function on podosome arrangement is dependent on a functional GEF domain (Gringel et al, 2006). GIT proteins (GIT1 and GIT2) possess Arf GAP activity and can bind to paxillin.…”

Actopaxin (α-Parvin) Phosphorylation Is Required for Matrix Degradation and Cancer Cell Invasion

“…Indeed, addition of myosin inhibitors rescued matrix degradation in Quint actopaxin U2OS cells as well as cell invasion of Quint actopaxin MDA-MB-231 cells. Conversely, both the Cdc42/Rac1 GEF, ␤-PIX, and its effector and binding partner PAK1 have previously been reported to be required for invadopodia formation and function, but little is known about the mechanism regulating their activity in this context (28,(43)(44)(45). Actopaxin binding to ␤-PIX provides a new mechanism by which ␤-PIX and PAK1 may be recruited to adhesions to coordinate their turnover during migration and/or transformation to invasive structures.…”