Mary Ann Sells

Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1.

The stress-activated p38 mitogen-activated protein (MAP) kinase defines a subgroup of the mammalian MAP kinases that appear to play a key role in regulating inflammatory responses. Co-expression of constitutively active forms of Rac and Cdc42 leads to activation of p38 while dominant negative Rac and Cdc42 inhibit the ability of interleukin-1 to increase p38 activity. p21-activated kinase 1 (Pak1) is a potential mediator of Rac/Cdc42 signaling, and we observe that Pak1 stimulates p38 activity. A dominant negative Pak1 suppresses both interleukin-1- and Rac/Cdc42-induced p38 activity. Rac and Cdc42 appear to regulate a protein kinase cascade initiated at the level of Pak and leading to activation of p38 and JNK.

Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells.

BACKGROUND The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. Recently, the serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. RESULTS Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton. CONCLUSIONS These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility.

p21-Activated Kinase 1 Phosphorylates the Death Agonist Bad and Protects Cells from Apoptosis

ABSTRACT Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-xL. Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-xL and its association with 14-3-3τ. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B fromClostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-xL and the increased association of Bad with 14-3-3τ. Our findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways.

Emerging from the Pak: the p21-activated protein kinase family.

The p21-activated protein kinases (PAKs) are members of a growing family of regulatory enzymes that may play roles in diverse phenomena such as cellular morphogenesis, the stress response and the pathogenesis of AIDS. PAKs were initially discovered as binding partners for small (21 kDa) GTPases that regulate actin polymerization, and recent evidence has shown that some members of the PAK family may be effectors for related GTPases that are involved in intracellular vesicle trafficking. Because the downstream signalling pathways for all such GTPases are poorly understood, intense studies are under way to discern the role of PAK and its cousins. In this review, the authors highlight some of the established properties of the extended PAK family and discuss current controversies regarding their possible roles as GTPase effectors.

Fission yeast pak1+ encodes a protein kinase that interacts with Cdc42p and is involved in the control of cell polarity and mating.

A STE20/p65pak homolog was isolated from fission yeast by PCR. The pak1+ gene encodes a 72 kDa protein containing a putative p21‐binding domain near its amino‐terminus and a serine/threonine kinase domain near its carboxyl‐terminus. The Pak1 protein autophosphorylates on serine residues and preferentially binds to activated Cdc42p both in vitro and in vivo. This binding is mediated through the p21 binding domain on Pak1p and the effector domain on Cdc42p. Overexpression of an inactive mutant form of pak1 gives rise to cells with markedly abnormal shape with mislocalized actin staining. Pak1 overexpression does not, however, suppress lethality associated with cdc42‐null cells or the morphologic defeat caused by overexpression of mutant cdc42 alleles. Gene disruption of pak1+ establishes that, like cdc42+, pak1+ function is required for cell viability. In budding yeast, pak1+ expression restores mating function to STE20‐null cells and, in fission yeast, overexpression of an inactive form of Pak inhibits mating. These results indicate that the Pak1 protein is likely to be an effector for Cdc42p or a related GTPase, and suggest that Pak1p is involved in the maintenance of cell polarity and in mating.

Protein tyrosine phosphatase 1B negatively regulates integrin signaling

Protein tyrosine phosphatase (PTP) 1B has long been known to regulate cell proliferation negatively, but the mechanism by which this inhibition occurs is poorly defined. We have shown previously that PTP1B binds to, and dephosphorylates, p130(Cas) (Crk-associated substrate) [1], a protein that is thought to play a role in integrin signaling [2,3]. In this report, we present evidence that PTP1B interferes specifically with cell-adhesion-stimulated, but not growth-factor-stimulated, signaling pathways. In rat fibroblasts that overexpress PTP1B, the activation of mitogen-activated protein (MAP) kinase by growth factors was not affected, but activation by cell adhesion was markedly impaired. The inhibition of adhesion-dependent MAP kinase activation by PTP1B required an intact proline-rich region in the carboxyl terminus of PTP1B, a region we have shown to mediate binding to the Src-homology 3 (SH3) domain of p130Cas [1]. Overexpression of wild-type PTP1B, but not of a proline-to-alanine mutant form (PA-PTP1B) that is unable to bind or dephosphorylate p130Cas, interfered with cell spreading, cytoskeletal architecture, and the formation of focal adhesion complexes. Cells overexpressing wild-type PTP1B also displayed markedly reduced migration in response to a fibronectin gradient, whereas cells expressing the PA-PTP1B mutant migrated normally. These data indicate that PTP1B exerts its inhibitory effects via proline-dependent interactions with one or more critical components of the adhesion-dependent signaling apparatus, and suggest that one of these components may be p130Cas.

P21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics

Mast cells are key participants in allergic diseases via activation of high-affinity IgE receptors (FcepsilonRI) resulting in release of proinflammatory mediators. The biochemical pathways linking IgE activation to calcium influx and cytoskeletal changes required for intracellular granule release are incompletely understood. We demonstrate, genetically, that Pak1 is required for this process. In a passive cutaneous anaphylaxis experiment, W(sh)/W(sh) mast cell-deficient mice locally reconstituted with Pak1(-/-) bone marrow-derived mast cells (BMMCs) experienced strikingly decreased allergen-induced vascular permeability compared with controls. Consistent with the in vivo phenotype, Pak1(-/-) BMMCs exhibited a reduction in FcepsilonRI-induced degranulation. Further, Pak1(-/-) BMMCs demonstrated diminished calcium mobilization and altered depolymerization of cortical filamentous actin (F-actin) in response to FcepsilonRI stimulation. These data implicate Pak1 as an essential molecular target for modulating acute mast cell responses that contribute to allergic diseases.

Cell cycle-regulated phosphorylation of p21-activated kinase 1.

Mammalian p21-activated kinase 1 (Pak1) is a highly conserved effector for the small GTPases Cdc42 and Rac1. In lower eukaryotes, Pak1 homologs are regulated during the cell cycle by phosphorylation. Here, we show that Pak1 is phosphorylated during mitosis in mammalian fibroblasts. This phosphorylation occurs at a single site, Thr 212, within a domain that is unique to Pak1. Cdc2 phosphorylates Pak1 at the identical site in vitro, and inhibition of Cdc2 abolishes Pak1 mitotic phosphorylation in vivo, indicating that Cdc2 is the kinase responsible for phosphorylating Pak1 in mitotic cells. Expression of a Pak1 mutant in which Thr 212 is replaced with a phosphomimic (aspartic acid) has marked effects on the rate and extent of postmitotic spreading of fibroblasts. The mitotic phosphorylation of Pak1 does not alter the basal or Rac-stimulated activity of this kinase, but it does affect the coimmunoprecipitation of at least three proteins with Pak1. These findings are the first to implicate a mammalian Pak in cell cycle regulation and suggest that Pakl, as a result of phosphorylation by Cdc2, alters its association with binding partners and/or substrates that are relevant to the morphologic changes associated with cell division.

Transformation Suppression by Protein Tyrosine Phosphatase 1B Requires a Functional SH3 Ligand

ABSTRACT We have recently shown that protein tyrosine phosphatase 1B (PTP1B) associates with the docking protein p130Cas in 3Y1 rat fibroblasts. This interaction is mediated by a proline-rich sequence on PTP1B and the SH3 domain on p130Cas. Expression of wild-type PTP1B (WT-PTP1B), but not a catalytically competent, proline-to-alanine point mutant that cannot bind p130Cas(PA-PTP1B), causes substantial tyrosine dephosphorylation of p130Cas (F. Liu, D. E. Hill, and J. Chernoff, J. Biol. Chem. 271:31290–31295, 1996). Here we demonstrate that WT-, but not PA-PTP1B, inhibits transformation of rat 3Y1 fibroblasts by v-crk, -src, and -ras, but not by v-raf. These effects on transformation correlate with the phosphorylation status of p130Cas and two proteins that are associated with p130Cas, Paxillin and Fak. Expression of WT-PTP1B reduces formation of p130Cas-Crk complexes and inhibits mitogen-activated protein kinase activation by Src and Crk. These data show that transformation suppression by PTP1B requires a functional SH3 ligand and suggest that p130Cas may represent an important physiological target of PTP1B in cells.

Epitope-tag vectors for eukaryotic protein production

We report on the construction and use of two eukaryotic expression vectors which add well-characterized epitope tags to the N termini of proteins. The utility of these vectors is demonstrated for detecting the expression of a variety of proteins. As the addition of these epitope tags can in some cases obviate the need to generate specific antisera to each individual protein, these vectors provide a facile means both to monitor protein expression and to purify such expressed proteins.