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.

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 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.

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.

Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast.

p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined. We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity. Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe. Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity. A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains. These results show that S. pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity.

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.

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.

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.

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.

Interaction of the Nck adapter protein with p21-activated kinase (PAK1).

The p21-activated kinases (PAKs) link G protein-coupled receptors and growth factor receptors (S. Dharmawardhane, R. H. Daniels, and G. M. Bokoch, submitted for publication) to activation of MAP kinase cascades and to cytoskeletal reorganization (M. A. Sells, U. G. Knaus, D. Ambrose, S. Bagrodia, G. M. Bokoch, and J. Chernoff, submitted for publication). The proteins that interact with PAK to mediate its cellular effects and to couple it to upstream receptors are unknown. We describe here a specific interaction of the Nck adapter molecule with PAK1 both in vitro and in vivo. PAK1 and Nck associate in COS-7 and Swiss 3T3 cells constitutively, but this interaction is strengthened upon platelet-derived growth factor receptor stimulation. We show that Nck binds to PAK1 through its second Src homology 3 (SH3) domain, while PAK1 interacts with Nck via the first proline-rich SH3 binding motif at its amino terminus. The interaction of active PAK1 with Nck leads to the phosphorylation of Nck at multiple sites. Association of Nck with PAK1 may serve to link this important regulatory kinase to cell activation by growth factor receptors.

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.

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.

Nerve growth factor stimulates tyrosine phosphorylation and activation of Src homology-containing protein-tyrosine phosphatase 1 in PC12 cells.

Rat PC12 cells respond to extracellular peptide growth factors in at least two distinct ways. When treated with nerve growth factor (NGF) PC12 cells exit the cell cycle and differentiate to a neuronal phenotype, whereas when treated with epidermal growth factor, they proliferate. We examined the potential role of Src homology 2 (SH2)-containing protein tyrosine phosphatases (PTPs) in the differentiation process. PC12 cells express substantial amounts of both SH-PTP1 and 2. SH-PTP1, but not SH-PTP2, becomes tyrosine phosphorylated following NGF, but not epidermal growth factor treatment. The enzymatic activity of SH-PTP1 toward an exogenous substrate following NGF treatment is increased 2-fold. We found that SH-PTP1 binds to the NGF receptor TrkA in vitro and that anti-TrkA immunoprecipitates have PTP activity. These results show that SH-PTP1 is differentially phosphorylated and activated by NGF in PC12 cells and suggest that this activation may play a role in NGF-induced differentiation.

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.

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.

Morphologic, immunohistochemical, and ultrastructural studies of the production of hepatitis B virus in vitro.

Well differentiated human hepatoblastoma Hep G2 cells after transfection with cloned hepatitis B virus (HBV) genomes produce replicative HBV DNA intermediates, high levels of HBsAg, HBeAg and HBcAg as well as mature Dane particles. To analyze the replication cycle of HBV, we studied the expression of HBV antigens with monoclonal antibodies by immunomorphologic methods in the transfected cells at various time intervals after plating. HBcAg and HBeAg were detected in the cytoplasm and less frequently in the nuclei of transfected cells. The percentage of positive cells increased with time after plating and reached a plateau of about 50% positive cells at 10 days. HBsAg and the large and middle HBsAg polypeptides were observed in the cytoplasm of transfected cells and a maximum of 20 to 30% positive cells was reached during the 3rd week after plating. Examination of viable cells in suspension revealed HBcAg/HBeAg and HBsAg expression on the cell surface. Electron microscopy demonstrated characteristic core particles in the nuclei and cytoplasm and Dane particles in cytoplasmic vesicles and culture media of transfected cells. The HBV producing cells did not show any evidence of a cytopathic effect. These observations demonstrate significant similarities between the HBV DNA transfected cells and infected human hepatocytes which support active HBV replication in vivo. Taken together, the results suggest that the cultured cells may serve as a model to elucidate a number of unsolved problems of the molecular and cellular pathobiology of hepatitis B.

Replicative intermediates of hepatitis B virus in HepG2 cells that produce infectious virions.

Clonal cells derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete hepatitis B surface antigen particles, nucleocapsids, and virions (M. A. Sells, M.-L. Chen, and G. Acs, Proc. Natl. Acad. Sci. USA 84:1005-1009, 1987) which elicit acute hepatitis in chimpanzees (G. Acs, M. A. Sells, R. H. Purcell, P. Price, R. Engle, M. Shapiro, and H. Popper, Proc. Natl. Acad. Sci. USA 84:4641-4644, 1987). We report here the initial characterization of the viral nucleic acids produced in this culture system. Kinetic analyses of nuclear, cytoplasmic, and extracellular HBV DNAs were performed by Southern blotting with radiolabeled HBV strand-specific probes. The results from these analyses indicate that at the stationary cellular growth phase, there is a dramatic increase in the rate at which HBV DNA accumulates. Incomplete double- and single-stranded forms of the HBV genome were detected in the nuclear and cytoplasmic fractions as well as in the extracellular medium. In addition, the nuclear DNA apparently includes multiple complete copies of the HBV genome chromosomally integrated and full-length covalently closed circular HBV DNA. Multiple HBV-specific polyadenylated RNAs with lengths of 3.5, 2.5, and 2.1 kilobases were identified by Northern (RNA) blot analysis. S1 nuclease mapping and primer extension identified a single 3' end and multiple unique initiation sites corresponding to nucleotides just 5' to the pre-S1 region, as well as upstream and within the pre-S2 and precore regions. The nucleic acid profile obtained from these analyses is essentially a facsimile of that obtained by studying liver tissue from HBV-infected individuals.

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.