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 ofone 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.The human pathogen hepatitis B virus (HBV) is one of a family of small DNA hepadnaviruses that share the ability to cause liver damage but differ completely in their host range specificity. The genome of HBV (as well as those of all of the hepadnaviruses) is a relaxed circular, partially double-stranded DNA molecule that is held together by hydrogen bonding of the ='300-base-pair (bp) 5' cohesive termini (1). The (-) strand has an invariable length of -3.2 kilobases (kb), whereas the (+) strand is .20% of this length (2).Investigation of the expression and replication of the HBV genome as well as the full viral life cycle has been hampered by the lack of an in vitro tissue culture system in which HBV is propagated. In numerous attempts to rectify this situation, several mammalian cell lines have been transfected with cloned HBV DNA (3-10). Thus Knowles. These cells were transfected with pDolTHBV-1, a vector that contains two head-to-tail dimers of HBV in a tail-to-tail orientation. Transformation with the plasmid was mediated by exposure of the cells to pDolTHBV-1 in the presence of 5 umg of Polybrene per ml (17,18). The cultures were incubated at 37°C for 6 hr, and the cells then were shocked for 4 min with 30% dimethyl sulfoxide in minimal essential medium (MEM), washed several times with phosphate-buffered saline (PBS) containing Ca2' and Mg2+, and maintained thereafter in MEM supplemented with 10% fetal bovine serum and 380 ,ug of G418 per ml. Clones of cells that grew in the presence of G418 were isolated, allowed to grow to confluence, and tested for their ability to synthesize and secrete HBsAg and HBeAg. One of the clones obtained in this manner, designated 2.2.15, has been maintained for >6 months and was analyzed as described below. All cul...

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.

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.

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.

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 5Ј-triphosphatases and have been shown to regulate the actin-myosin cytoskeleton. In fibroblasts PAK1 localizes to areas of membrane ruffling, as well as to amiloridesensitive 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 plateletderived 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. Introductionp21-activated kinases (PAKs 1, 2, 3) are a group of 62-68-kDa serine/threonine kinases originally identified as targets of the small guanosine 5Ј-triphosphatases (GTPases) Rac and Cdc42 (Manser et al., 1994;Knaus and Bokoch, 1998). GTP-bound forms of Rac and Cdc42 stimulate the activity of PAKs by binding to a specific N-terminal p21-binding site (Manser et al., 1994;Burbelo et al., 1995;, thereby relieving the influence of an adjacent kinase autoinhibitory domain (Zhao et al., 1998;Zenke et al., 1999). PAKs have been implicated as effectors in several Racand Cdc42-regulated signaling pathways, including regulation of the phagocyte NADPH oxidase , p38 and C-jun N-terminal kinase activation (Zhang et al., 1995;Bagrodia et al., 1995;Tang et al., 1997), and modulation of the actin cytoskeleton (Dharmawardhane et al., 1997;Manser et al., 1997;Daniels et al., 1998).Introduction of a constitutively active PAK1 (H83,86L) N-terminal mutant into a variety of adherent cells induces the formation of cortical actin structures similar to those known to be regulated by Rac and Cdc42, including membrane ruffles and lamellipodia, filopodia, and focal complexes (Dharmawardhane et al., 1997;M...

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.

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting

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