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.

Temporal and spatial distribution of activated Pak1 in fibroblasts.

p21-activated kinases (Paks) are effectors of the small GTPases Cdc42 and Rac, and are thought to mediate some of the cytoskeletal and transcriptional activities of these proteins. To localize activated Pak1 in cells, we developed an antibody directed against a phosphopeptide that is contained within the activation loop of Pak1. This antibody specifically recognizes the activated form of Pak1. Immunofluorescence analysis of NIH-3T3 cells coexpressing activated Cdc42 or Rac1 plus wild-type Pak1 shows that activated Pak1 accumulates at sites of focal adhesion, throughout filopodia and within the body and edges of lamellipodia. Platelet-derived growth factor stimulation of NIH-3T3 cells shows a pattern of Pak1 activation similar to that observed with Rac1. During closure of a fibroblast monolayer wound, Pak1 is rapidly activated and localizes to the leading edge of motile cells, then gradually tapers off as the wound closes. The activation of Pak1 by wounding is blocked by inhibitors of phosphatidylinositol 3-kinase, and Src family kinases, but not by an inhibitor of the epidermal growth factor receptor. These findings indicate that activated Pak1, and by extension, probably activated Cdc42 or Rac, accumulates at sites of cortical actin remodeling in motile fibroblasts.

p21-activated kinase 1 (Pak1) regulates cell motility in mammalian fibroblasts.

The p21 (Cdc42/Rac) activated kinase Pak1 regulates cell morphology and polarity in most, if not all, eukaryotic cells. We and others have established that Pak's effects on these parameters are mediated by changes in the organization of cortical actin. Because cell motility requires polarized rearrangements of the actin/myosin cytoskeleton, we examined the role of Pak1 in regulating cell movement. We established clonal tetracycline-regulated NIH-3T3 cell lines that inducibly express either wild-type Pak1, a kinase-dead, or constitutively-active forms of this enzyme, and examined the morphology, F-actin organization, and motility of these cells. Expression of any of these forms of Pak1 induced dramatic changes in actin organization which were not inhibited by coexpression of a dominant-negative form of Rac1. Cells inducibly expressing wild-type or constitutively-active Pak1 had large, polarized lamellipodia at the leading edge, were more motile than their normal counterparts when plated on a fibronectin-coated surface, and displayed enhanced directional movement in response to an immobilized collagen gradient. In contrast, cells expressing a kinase-dead form of Pak1 projected multiple lamellipodia emerging from different parts of the cell simultaneously. These cells, though highly motile, displayed reduced persistence of movement when plated on a fibronectin-coated surface and had defects in directed motility toward immobilized collagen. Expression of constitutively activated Pak1 was accompanied by increased myosin light chain (MLC) phosphorylation, whereas expression of kinase-dead Pak1 had no effect on MLC. These results suggest that Pak1 affects the phosphorylation state of MLC, thus linking this kinase to a molecule that directly affects cell movement.

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.

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.

Transformation suppression by protein tyrosine phosphatase 1B requires a functional SH3 ligand.

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.

p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis.

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-x(L). Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x(L) and its association with 14-3-3tau. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium 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-x(L) and the increased association of Bad with 14-3-3tau. 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.

Regulation of macropinocytosis by p21-activated kinase-1.

The process of macropinocytosis is an essential aspect of normal cell function, contributing to both growth and motile processes of cells. p21-activated kinases (PAKs) are targets for activated Rac and Cdc42 guanosine 5'-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloride-sensitive pinocytic vesicles. Expression of a PAK1 kinase autoinhibitory domain blocked both platelet-derived growth factor- and RacQ61L-stimulated uptake of 70-kDa dextran particles, whereas an inactive version of this domain did not, indicating that PAK kinase activity is required for normal growth factor-induced macropinocytosis. The mechanisms by which PAK modulate macropinocytosis were examined in NIH3T3 cell lines expressing various PAK1 constructs under the control of a tetracycline-responsive transactivator. Cells expressing PAK1 (H83,86L), a mutant that dramatically stimulates formation of dorsal membrane ruffles, exhibited increased macropinocytic uptake of 70-kDa dextran particles in the absence of additional stimulation. This effect was not antagonized by coexpression of dominant-negative Rac1-T17N. In the presence of platelet-derived growth factor, both PAK1 (H83,86L) and a highly kinase active PAK1 (T423E) mutant dramatically enhanced the uptake of 70-kDa dextran. Neither wild-type PAK1 nor vector controls exhibited enhanced macropinocytosis, nor did PAK1 (H83,86L) affect clathrin-dependent endocytic mechanisms. Active versions of PAK1 enhanced both growth factor-stimulated 70-kDa dextran uptake and efflux, suggesting that PAK1 activity modulated pinocytic vesicle cycling. These data indicate that PAK1 plays an important regulatory role in the process of macropinocytosis, perhaps related to the requirement for PAK in directed cell motility.

Isolation of oval cells and transitional cells from the livers of rats fed the carcinogen DL-ethionine.

For the characterization of the metabolic and biologic properties of oval cells (i.e., cells emerging in the livers of rats treated with chemical carcinogens due to proliferation of bile ductular and/or duct cells) and transitional cells (i.e., cells having properties intermediate between those of oval cells and hepatocytes), these cells were isolated from the livers of Sprague-Dawley rats fed DL-ethionine for 4-5 weeks. The livers were dissociated into single cells by perfusion in situ with collagenase, and total cell suspensions were allowed to stand at unit gravity for 10 minutes to separate parenchymal (hepatocytes) from nonparenchymal cells. Nonparenchymal cells were centrifuged in linear gradients of Metrizamide (8-24% wt/vol), and 2-ml fractions were collected from the gradients. The cells in the fractions were defined by light microscopy, electron microscopy, and histochemical and immunofluorescence methods. A cell isolate was thus obtained consisting of Kupffer's cells (approximately 20%), bile ductular and/or duct cells and oval cells (approximately 30%), and transitional cells (approximately 50%). A twofold enrichment of bile ductular and/or duct cells and their derivatives was achieved over that found in the nonparenchymal cell fraction before isopyknic gradient centrifugation.

Hepatocarcinogenic and promoting action of a choline-devoid diet in the rat.

Male Fisher 344 rats were solely fed a choline-supplemented diet for 65 to 105 weeks or a choline-devoid diet for 24 to 102 weeks. Hepatocellular carcinomas developed in the latter animals, beginning at 24 weeks. Other groups of rats were given a single dose of 20 mg diethylnitrosamine/kg, 18 h after a partial hepatectomy and were fed, 4 weeks thereafter, either a choline-supplemented, or a choline-devoid diet for up to 48 weeks. In rats fed the choline-supplemented diet, the only relevant lesion observed was a small transect number of foci of enzyme-altered hepatocytes. On the other hand, a significant number of foci, of preneoplastic nodules, and of hepatocellular tumors developed in rats fed the choline-devoid diet. The results obtained are consistent with those previously reported by others, indicating that diets devoid of choline, or of choline and methionine, are carcinogenic. The diets appear to act as complete carcinogens, since they are also efficient promoters of chemical hepatocarcinogenesis, as shown again, in the present study, by the results obtained in the diethylnitrosamine-pretreated rats.

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.

Delivery of protein into cells using polycationic liposomes.

We describe a procedure for delivery of purified proteins into a variety of tissue culture cells using a new polycationic lipid preparation, LipofectAMINE. Several different proteins, with diverse physical properties, can be delivered into cells by this method. Compared with commercially available monocationic lipids, protein delivery using LipofectAMINE is more efficient. Unlike other methods for protein delivery, the lipofection procedure is simple, inexpensive and effective for both adherent and nonadherent cell types. Proteins introduced into cells using this method are shown to be biochemically and biologically active.

Characterization of RNA transcripts and virally coded proteins synthesized in mouse fibroblasts transfected with hepatitis B DNA: HBeAg synthesis in HBcAg-negative cells with active core-antigen genes.

A line of mouse cells expressing hepatitis B virus (HBV) surface and 'e' antigens identical in their physico-chemical properties to antigens from patients infected with HBV was isolated after transfection of 3T3 cells with cloned HBV DNA. The studies reported here indicate that the cells contain uninterrupted copies of the entire HBV genome which are unmethylated on CCGG sites and have no gross deletions or rearrangements. The entire core region is transcribed into polyadenylated RNAs large enough to serve as messengers for production of viral core antigen (HBcAg) yet no HBcAg can be detected. This suggests that the cells produce a primary translation product copied from the HBcAg messenger which either cannot assume the proper configuration for display of HBcAg determinants or is rapidly converted to HBeAg by proteolysis.

Developing self-esteem in urban youth.

A group of Black, urban, teenage males from the Central Detroit catchment area were certified through the Comprehensive Employment and Training Act's (CETA) Summer Youth Employment Program to participate in a Youth and Self-Esteem project. The project's objective was to expose the participants to older Black males who have made significant contributions to community. Through research and direct interviews, we hoped to identify the coping skills which enabled them to survive and excel and share these characteristics and strategies with the youth of today.

Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.

The hepatoblastoma cell line Hep G2 was transfected with a plasmid carrying the gene that confers resistance to G418 and four 5'-3' tandem copies of the hepatitis B virus (HBV) genome positioned such that two dimers of the genomic DNA are 3'-3' with respect to one another. Cells of one clone that grew in the presence of G418 produce high levels of hepatitis B e antigen and of hepatitis B surface antigen. HBV DNA is carried by these cells as chromosomally integrated sequences and episomally as relaxed circular, covalently closed, and incomplete copies of the HBV genome. Viral DNA was detected also in conditioned growth medium at the buoyant densities characteristic for infectious Dane and immature core particles. Finally, HBV-specific components morphologically identical to the 22-nm spherical and filamentous hepatitis B surface antigen particles as well as 42-nm Dane particles were visualized by immunoelectron microscopic analysis. Therefore, we have demonstrated that the Hep G2 cell line can support the assembly and secretion not only of several of the replicative intermediates of HBV DNA but also of Dane-like particles. This in vitro system can now be used to study the life cycle of HBV and the reaction of immunocompetent cells with cells carrying HBV.

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin.

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.

Replicative intermediate of hepatitis B virus in transfected murine fibroblasts.

The NIH 3T3-derived cell line psi AM22b, which carries a defective Moloney murine leukemia virus, was transfected with a plasmid carrying the neo gene and two head-to-tail copies of the hepatitis B virus (HBV) genome positioned with opposing polarities. Both the two HBV dimers and the neo gene were located between two Moloney murine leukemia virus long terminal repeats. Poly(A)+ RNAs isolated from one clone that grew in the presence of G418 contained the two major classes of HBV-specific transcripts (3.5-kilobase pregenome and 2.1-kilobase mRNAs) in approximately equivalent amounts, which was reminiscent of the profiles of viral mRNAs from the livers of infected humans and chimpanzees.