A GTPASE-INDEPENDENT MECHANISM OF P21-ACTIVATED KINASE ACTIVATION - REGULATION BY SPHINGOSINE AND OTHER BIOLOGICALLY-ACTIVE LIPIDS

p21-activated kinases (PAKs) are serine/threonine kinases that have be en identified as targets for the small GTPases Rac and Cdc42. PAKs hav e been implicated in cytoskeletal regulation, stimulation of mitogen-a ctivated protein kinase cascades, and in control of the phagocyte NADP H oxidase. Membrane targeting of PAK1 induced increased kinase activit y in a GTPase-independent manner, suggesting that other mechanisms for PAK regulation exist. We observed concentration- and time-dependent a ctivation of PAK1 by sphingosine and several related long chain sphing oid bases but not by ceramides or a variety of other lipids, Although phospholipids were generally ineffective, phosphatidic acid and phosph atidylinositol also had stimulatory effects on PAK1, Lipid stimulation induced a similar level of PAK1 activity as did stimulation by GTPase s, and the patterns of PAK1 autophosphorylation determined after parti al tryptic digestion and two-dimensional peptide analysis were similar with each class of activator. Lipid stimulation of PAK1 activity was dependent upon intact PAK kinase activity, as indicated by studies wit h a kinase-dead PAK1 mutant, Treatment of COS-7 cells expressing wild type PAK1 with sphingosine, fumonisin B, or sphingomyelinase, all of w hich are able to elevate the levels of free sphingosine, induced incre ased activity of PAK1 as determined using a p47(phox) peptide substrat e, Studies using PAK1 mutants suggest that lipids act at a site overla pping or identical to the GTPase-binding domain on PAK. The inactive s phingosine derivative N,N-dimethylsphingosine was an effective inhibit or of PAK1 activation in response to either sphingosine or Cdc42, Our results demonstrate a novel GTPase-independent mechanism of PAK activa tion and, additionally, suggest that PAK(s) may be important mediators of the biological effects of sphingolipids.