Expression of Constitutively Active α-PAK Reveals Effects of the Kinase on Actin and Focal Complexes

Abstract

G12V, which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause ␣-PAK autophosphorylation and kinase activation. Mapping ␣-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active ␣-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42 G12V or Rac1 G12V. Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.

Novel small GTPase M-Ras participates in reorganization of actin cytoskeleton

“…Transfection of Myc-tagged constitutively active Cdc42 G12V to C3H/10T1/2 cells also induced actin ®lament-containing microspikes within 18 h (data not shown). These cells became round and lost stress ®bers as has been shown by Manser et al (1997) but never exhibited the arborized forms even 48 h after the transfection (data not shown). Figure 5 GDP/GTP-binding and GTPase assay.…”

“…The peripheral long processes of the actin focicontaining cells and the cortical microspikes of the arborized cells, however, seem to correspond to the retraction ®bers because these cell bodies appeared to be contracted. In addition to the microspike formation, Cdc42 also induces loss of stress ®bers and formation of the actin-containing podosome-like structures, which are more evident in the transfected or the cDNA-injected cells (Kozma et al, 1995;Dutartre et al, 1996;Manser et al, 1997). The actin foci and disassembly of actin stress ®bers in the MRas-injected cells might be equivalent to the podosome-like structures and loss of stress ®bers induced by Cdc42.…”

“…It remains to be examined whether the actin foci-containing cells with long processes are in a transitional state to the arborized cells or whether the actin foci-containing cells are generated only under the serum-starved conditions. Cdc42 induces peripheral actin microspikes not only by extension (®lopodia) but also by cell retraction (retraction ®bers) (Kozma et al, 1995;Manser et al, 1997). Since the ®lopodia and the retraction ®bers are indistinguishable on the basis of phalloidin staining, time-lapse microscopy is required to determine the type of microspikes induced by MRas.…”

“…The actin foci and disassembly of actin stress ®bers in the MRas-injected cells might be equivalent to the podosome-like structures and loss of stress ®bers induced by Cdc42. Manser et al (1997) have shown that microinjection of the cDNA encoding the Cdc42 e ector a-PAK fused with Cdc42 G12V into HeLa cells give rise to collapsed cell shapes with radiating processes. The arborized cell ®gures induced by the transfection of M-Ras G22V are reminiscent of these collapsed cell shapes.…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…The GIT-PIX-PAK complex appears to constitute a functional, tripartite module in which GIT targets both PIX and PAK to focal adhesions, focal complexes or synapses [64,66,67]. Early models suggested that PIX activated Rac and/or Cdc42, which then activated PAK [68]. Active PAK would then stimulate focal adhesion turnover and extension of lamellipodia, thereby promoting cell migration [67,69].…”