PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway

Abstract

The serine/threonine kinase Raf-1 acts downstream of Ras in the MAPK pathway leading to ERK activation in response to mitogens. Raf-1 has oncogenic potential, but is normally controlled by a complex interplay of inhibitory and activating mechanisms. Although Raf-1 is phosphorylated in unstimulated cells, mitogens cause its membrane recruitment by Ras and subsequent phosphorylation on additional sites. Some of these events modulate Raf-1 kinase activity while others determine interactions with other proteins. These changes regulate the ability of Raf-1 to phosphorylate its downstream targets MEK1 and MEK2. Rho family small G proteins act synergistically with Raf-1 to stimulate the ERK pathway by a cross-cascade mechanism that enhances MEK phosphorylation by Raf-1. Here we show that both Raf-1 and MEK1 are phosphorylated by PAK1 and that mutations at PAK1 phosphorylation sites in either protein prevent cross-cascade activation. In contrast, MEK1 activation by constitutively-active Raf-1 is refractory to mutations at PAK1 phosphorylation sites. Phosphorylation of MEK1 on serine 298 does not appear to regulate the interaction between Raf-1 and MEK1, but rather the ability of Raf-1 to phosphorylate MEK1 with which it is complexed in vivo. Our ®ndings indicate that PAK1 primes MEK1 for activation by Raf-1 and imply another level of regulation in the ERK cascade.

P-21-activated protein kinase-1 functions as a linker between insulin and Wnt signaling pathways in the intestine

“…PAK-1 may also phosphorylate MEK1 at Ser298 to prime MEK1 for its subsequent activation by Raf-1 ( Coles and Shaw, 2002;Park et al, 2007). We observed that PAK-1 knockdown significantly reduced both basal and insulin-stimulated ERK1/2 phosphorylation.…”

“…As MEK has been identified as one of the downstream targets of PAK-1 (Coles and Shaw, 2002;Eblen et al, 2002;Slack-Davis et al, 2003;Park et al, 2007), we next examined whether the MEK-ERK signaling pathway participates in insulin-stimulated c-Myc expression and in b-cat nuclear content and function. PD98059 is a widely used inhibitor of MEK (Alessi et al, 1995;Dudley et al, 1995).…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…Thus, alternative Ras-independent mechanisms may activate ERK during adhesion. In this regard, we and others have investigated the role of the small GTPases Rac and Cdc42 and their downstream effector PAK in activating ERK signaling during adhesion [16,17]. We and others have reported that c-Src and FAK dependent PAK phosphorylation of MEK1 may prime the ERK module for activation by low levels of Raf activity during adhesion [18], although it is possible that PAK may directly activate MEK1 in a Raf-independent fashion [19].…”

Experimental therapeutic approaches to peripheral nerve tumors

“…128 Merlin, which has also recently been shown to be stabilized by the presence of an adapter protein, NGB, 76 is inactivated through Rac/Pak-mediated signaling by phosphorylation. 22 In contrast, activation through dephosphorylation is at least in part achieved through the action of MYPT-1-PP1-␦, a myosin phosphatase. 61 Myosin phosphatase activity is stimulated in part through CD44 transmembrane protein activation, providing a mechanism by which cell-cell contact may achieve growth inhibition.…”

“…Interplay between Ras-and Rac-mediated signaling underscores the importance of merlin function at the levels of Ras and Rac. The Ras/Raf-mediated activation of downstream Mek kinases requires prior phosphorylation by Rac, 22 which may be activated by either Ras-dependent or Rasindependent mechanisms. Inhibition of Rac/Pak signaling has also been demonstrated through merlin-mediated inactivation of PI3K.…”