p21-activated kinases (Paks) are a highly conserved family of enzymes that bind to and are activated by small GTPases of the Cdc42 and Rac families. With the notable exception of plants, nearly all eukaryotes encode one or more Pak genes, indicating an ancient origin and important function for this family of enzymes. Genetic approaches in many different experimental systems, ranging from yeast to mice, are beginning to decipher the different functions of Paks. Although some of these functions are unique to a given organism, certain common themes have emerged, such as the activation of mitogen-activated protein kinase (MAPK) cascades and the regulation of cytoskeletal structure through effects on the actin and tubulin cytoskeletons.

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The canonical extracellular-regulated kinase (ERK) signaling cascade, consisting of the Ras-Raf-Mek-ERK module, is critically important to many cellular functions. Although the general mechanism of activation of the ERK cascade is well established, additional noncanonical components greatly influence the activity of this pathway. Here, we focus on the group A p21-activated kinases (Paks), which have previously been implicated in regulating both c-Raf and Mek1 activity, by phosphorylating these proteins at Ser

The US3 protein is a viral serine/threonine kinase that is conserved among all members of the Alphaherpesvirinae. The US3 protein of different alphaherpesviruses causes dramatic alterations in the actin cytoskeleton, such as the disassembly of actin stress fibers and formation of cell projections, which have been associated with increased intercellular virus spread. Here, we find that inhibiting group A p21-activated kinases (PAKs), which are key regulators in Cdc42/Rac1 Rho GTPase signaling pathways, impairs US3-mediated actin alterations. By using PAK1 ؊/؊ and PAK2 ؊/؊ mouse embryo fibroblasts (MEFs), we show that US3-mediated stress fiber disassembly requires PAK2, whereas US3-mediated cell projection formation mainly is mediated by PAK1, also indicating that PAK1 and PAK2 can have different biological effects on the organization of the actin cytoskeleton. In addition, US3 was found to bind and phosphorylate group A PAKs. Lack of group A PAKs in MEFs was correlated with inefficient virus spread. Thus, US3 induces its effect on the actin cytoskeleton via group A PAKs.herpesvirus ͉ Rho GTPase signaling ͉ stress fibers ͉ projections ͉ viral kinase A lphaherpesviruses constitute the largest subfamily of the herpesviruses and contain different, closely related pathogens of humans and animals. In humans, herpes simplex virus (HSV) causes cold sores and genital lesions but may also cause keratitis, blindness, and encephalitis, and varicella-zoster virus (VZV) causes chickenpox and shingles. The closely related porcine alphaherpesvirus pseudorabies virus (PRV) is often used as a model organism to study general aspects of alphaherpesvirus biology (1).Recently, we and others have found that the US3 protein of PRV is able to reorganize the actin cytoskeleton of an infected host cell (2-6). This actin reorganization consists of the disassembly of actin stress fibers and the formation of actincontaining cell projections and is associated with an increase in the efficiency of intercellular virus spread (3). US3 is a serine/ threonine kinase that is conserved among all alphaherpesviruses, and US3 orthologs of HSV-2 and Marek's disease virus, a devastating alphaherpesvirus in poultry, have also been shown to induce disassembly of actin stress fibers (7,8). In addition, HSV-1 and VZV have also been reported to induce cell projections that may be involved in intercellular virus spread (9-11).These data on herpesviruses, as well as other data on other viruses, like retroviruses (e.g., HIV) and poxviruses (e.g., vaccina virus), have led to the recent concept that viral reorganizations of the cytoskeleton, including the formation of intercellular cell projections, present a novel and important route of viral transmission (12)(13)(14)(15)(16)(17). Elucidating the mechanism of these virusinduced cytoskeletal rearrangements may lead to novel avenues in the development of antiviral strategies.In the current report, we elucidate the mechanism of the US3-mediated effects on the actin cytoskeleton. We report that US3 induces the actin r...

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations in the NF1 locus, which encodes neurofibromin, a negative regulator of Ras. Patients with NF1 develop numerous neurofibromas, which contain many inflammatory mast cells that contribute to tumor formation. Subsequent to c-Kit stimulation, signaling from Ras to Rac1/2 to the MAPK pathway appears to be responsible for multiple hyperactive mast cell phenotypes; however, the specific effectors that mediate these functions remain uncertain. p21-activated kinase 1 (Pak1) is a downstream mediator of Rac1/2 that has been implicated as a positive regulator of MAPK pathway members and is a modulator of cell growth and cytoskeletal dynamics. Using an intercross of Pak 1 Ϫ/Ϫ mice with Nf1 ϩ/Ϫ mice, we determined that Pak1 regulates hyperactive Ras-dependent proliferation via a Pak1/ Erk pathway, whereas a Pak1/p38 pathway is required for the increased migration in Nf1 ϩ/Ϫ mast cells. Furthermore, we confirmed that loss of Pak1 corrects the dermal accumulation of Nf1 ϩ/Ϫ mast cells in vivo to levels found in wild-type mice. Thus, Pak1 is a novel mast cell mediator that functions as a key node in the MAPK signaling network and potential therapeutic target in NF1 patients. (Blood. 2008; 112:4646-4654) IntroductionMutations at the NF1 locus cause neurofibromatosis type 1 (NF1), a common human genetic disorder that affects approximately 1 in 3500 live births. 1 The NF1 gene encodes neurofibromin, a tumor suppressor that functions at least in part as a GTPase-activating protein (GAP) for p21 ras (Ras) 2 and accelerates the hydrolysis of active Ras-GTP to inactive NF1 is characterized by the development of tumors called neurofibromas, 4 which are composed of Schwann cells, fibroblasts, endothelial cells, and large numbers of degranulating mast cells. 5 Previous work in a genetically engineered murine model of NF1 that closely recapitulates the spontaneous tumor progression observed in patients has established that nullizygosity of Nf1 in tumorigenic Schwann cells is necessary but not sufficient for plexiform neurofibroma formation. 6 Furthermore, haploinsufficiency of Nf1 in at least a subset of lineages within the tumor microenvironment is required to promote neurofibroma development. 6 Besides mediating innate immune responses and allergic hypersensitivity, there is an emerging understanding that mast cells in the tumor microenvironment have relevance in the promotion of neoplastic development in multiple murine disease models. [7][8][9][10][11][12] We have previously shown that bone marrow-derived mast cells (BMMCs) haploinsufficient at Nf1 have hyperactivated Ras-GTP, as well as increases in Ras-dependent functions such as proliferation and migration after stem cell factor (SCF) stimulation. 13 Previous work has established that the excess proliferation in Nf1 ϩ/Ϫ BMMCs is the result of Ras activation of the class1A-PI-3 kinase (PI-3K) pathway. 14 Genetic and pharmacologic blockade of PI-3K activity in Nf1 ϩ/Ϫ cells leads to reduction of phosp...