Jonathan Chernoff

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.

Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin

The morphogenesis of dendritic spines, the major sites of excitatory synaptic transmission in the brain, is important in synaptic development and plasticity. We have identified an ephrinB-EphB receptor trans-synaptic signaling pathway which regulates the morphogenesis and maturation of dendritic spines in hippocampal neurons. Activation of the EphB receptor induces translocation of the Rho-GEF kalirin to synapses and activation of Rac1 and its effector PAK. Overexpression of dominant-negative EphB receptor, catalytically inactive kalirin, or dominant-negative Rac1, or inhibition of PAK eliminates ephrin-induced spine development. This novel signal transduction pathway may be critical for the regulation of the actin cytoskeleton controlling spine morphogenesis during development and plasticity.

p21-activated kinases: three more join the Pak.

The p21-activated kinases (Paks) are serine/threonine protein kinases that bind to and, in some cases, are stimulated by activated forms of the small GTPases, Cdc42 and Rac. With the recent discovery of several novel isoforms, Paks are now categorized into two subgroups based on architectural similarities. The Group I Paks (Pak1, Pak2, Pak3) have been studied in greater detail and shown to be involved in the regulation of cellular processes such as gene transcription, cell morphology, motility, and apoptosis. Here we summarize recent findings that shed light on the newly recognized Group II Paks (Pak4, Pak5, Pak6) and review both similarities and differences between kinases of the two Pak subgroups.

Caspase‐mediated activation and induction of apoptosis by the mammalian Ste20‐like kinase Mst1

Mst1 is a ubiquitously expressed serine–threonine kinase, homologous to the budding yeast Ste20, whose physiological regulation and cellular function are unknown. In this paper we show that Mst1 is specifically cleaved by a caspase 3‐like activity during apoptosis induced by either cross‐linking CD95/Fas or by staurosporine treatment. CD95/Fas‐induced cleavage of Mst1 was blocked by the cysteine protease inhibitor ZVAD‐fmk, the more selective caspase inhibitor DEVD‐CHO and by the viral serpin CrmA. Caspase‐mediated cleavage of Mst1 removes the C‐terminal regulatory domain and correlates with an increase in Mst1 activity in vivo, consistent with caspase‐mediated cleavage activating Mst1. Overexpression of either wild‐type Mst1 or a truncated mutant induces morphological changes characteristic of apoptosis. Furthermore, exogenously expressed Mst1 is cleaved, indicating that Mst1 can activate caspases that result in its cleavage. Kinase‐dead Mst1 did not induce morphological alterations and was not cleaved upon overexpression, indicating that Mst1 must be catalytically active in order to mediate these effects. Mst1 activates MKK6, p38 MAPK, MKK7 and SAPK in co‐transfection assays, suggesting that Mst1 may activate these pathways. Our findings suggest the existence of a positive feedback loop involving Mst1, and possibly the SAPK and p38 MAPK pathways, which serves to amplify the apoptotic response.

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.

Heregulin Regulates Cytoskeletal Reorganization and Cell Migration through the p21-activated Kinase-1 via Phosphatidylinositol-3 Kinase

The mechanisms through which heregulin (HRG) regulates the activities of breast cancer cells are currently unknown. We demonstrate that HRG stimulation of noninvasive breast cancer cells enhanced the conversion of globular to filamentous actin and the formation of membrane ruffles, stress fibers, filopodia, and lamellipodia and accompanied by increased cell migration. In addition, HRG triggered a rapid stimulation of p21-activated kinase1 (PAK1) activity and its redistribution into the leading edges of motile cells. The HRG-induced stimulation of PAK1 kinase activity followed phosphatidylinositol-3 kinase (PI-3 kinase) activation. Inhibition of PI-3 kinase activity blocked the activation of PAK1 kinase and also blocked cell migration in response to HRG. Furthermore, direct inhibition of PAK1 functions by the dominant-negative mutant suppressed the capacity of HRG to reorganize actin cytoskeleon structures. We also demonstrated that HRG stimulation promoted physical interactions between PAK1, actin, and human epidermal growth factor receptor 2 (HER2) receptors, and these interactions were dependent on the activation of PI-3 kinase. The blockade of HER2 receptor by an anti-HER2 monoclonal antibody resulted in the inhibition of HRG-mediated stimulation of PI-3 kinase/PAK pathway and also the formation of motile actin cytoskeleton structures but not extracellular signal-regulated kinases. These findings suggest a role of PI-3 kinase/PAK1-dependent reorganization of the cortical actin cytoskeleton in HRG-mediated increased cell migration, and these changes may have significant consequences leading to enhanced invasion by breast cancer cells.

An Isoform-Selective, Small-Molecule Inhibitor Targets the Autoregulatory Mechanism of p21-Activated Kinase

Autoregulatory domains found within kinases may provide more unique targets for chemical inhibitors than the conserved ATP-binding pocket targeted by most inhibitors. The kinase Pak1 contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. Pak1 activators relieve this autoinhibition and initiate conformational rearrangements and autophosphorylation events leading to kinase activation. We developed a screen for allosteric inhibitors targeting Pak1 activation and identified the inhibitor IPA-3. Remarkably, preactivated Pak1 is resistant to IPA-3. IPA-3 also inhibits activation of related Pak isoforms regulated by autoinhibition, but not more distantly related Paks, nor >200 other kinases tested. Pak1 inhibition by IPA-3 in live cells supports a critical role for Pak in PDGF-stimulated Erk activation. These studies illustrate an alternative strategy for kinase inhibition and introduce a highly selective, cell-permeable chemical inhibitor of Pak.

Regulation of PAK Activation and the T Cell Cytoskeleton by the Linker Protein SLP-76

Tyrosine phosphorylation of linker proteins enables the T cell antigen receptor (TCR)-associated protein tyrosine kinases to phosphorylate and regulate effector molecules that generate second messengers. We demonstrate here that the SLP-76 linker protein interacts with both nck, an adaptor protein, and Vav, a guanine nucleotide exchange factor for Rho-family GTPases. The assembly of this tri-molecular complex permits the activated Rho-family GTPases to regulate target effectors that interact through nck. In turn, assembly of this complex mediates the enzymatic activation of the p21-activated protein kinase 1 and facilitates actin polymerization. Hence, phosphorylation of linker proteins not only bridges the TCR-associated PTK, ZAP-70, with downstream effector proteins, but also provides a scaffold to integrate distinct signaling complexes to regulate T cell function.

Direct Binding of the Proline-rich Region of Protein Tyrosine Phosphatase 1B to the Src Homology 3 Domain of p130Cas

Protein tyrosine phosphatase 1B (PTP1B) is an abundant intracellular enzyme that is thought to act as a negative regulator of certain signaling pathways. The C terminus of PTP1B contains two proline-rich regions which conform to the canonical class II Src homology 3 domain binding motif, Pro-X-X-Pro-X-Arg. In this study, we establish that PTP1B interacts with Crk, Grb2, and p130Cas in vitro and with at least one of these, p130Cas, in intact cells. The interaction of PTP1B and p130Cas is independent of tyrosine phosphorylation but can be disrupted by replacing two critical proline residues in the proline-rich domain of PTP1B between amino acids 301 and 315. When wild-type PTP1B is expressed in 3Y1-v-crk cells, p130Cas shows substantial dephosphorylation, whereas the PTP1B proline mutant does not have this effect. In 3Y1 and 3Y1 v-crk-transformed fibroblasts, almost all of the total PTP1B and about 40% of total p130Cas co-sediment with membranes composed primarily of endoplasmic reticulum. These results suggest that the proline-rich domain between amino acids 301 and 315 in PTP1B binds Src homology 3-containing proteins and that p130Cas may be a physiological target of this phosphatase in cells.