ABSTRACT
Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.
Subject(s)
Actins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cell Cycle Proteins/physiology , G1 Phase/physiology , GTP-Binding Proteins/physiology , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases , Protein Kinases/physiology , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/physiology , Proteins/physiology , Signal Transduction/physiology , 3T3 Cells , Animals , COS Cells , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , DNA Replication , Enzyme Activation , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins , Integrins/metabolism , Mice , Mutagenesis, Site-Directed , Phosphorylation , Polymers , Proteins/genetics , Pseudopodia/ultrastructure , Recombinant Fusion Proteins/metabolism , Transfection , cdc42 GTP-Binding Protein , p21-Activated Kinases , ras GTPase-Activating ProteinsABSTRACT
BACKGROUND: The Rho-related GTP-binding proteins Cdc42 and Rac1 have been shown to regulate signaling pathways involved in cytoskeletal reorganization and stress-responsive JNK (Jun N-terminal kinase) activation. However, to date, the GTPase targets that mediate these effects have not been identified. PAK defines a growing family of mammalian kinases that are related to yeast Ste20 and are activated in vitro through binding to Cdc42 and Rac1 (PAK: p21 Cdc42-/Rac-activated kinase). Clues to PAK function have come from studies of Ste20, which controls the activity of the yeast mating mitogen-activated protein (MAP) kinase cascade, in response to a heterotrimeric G protein and Cdc42. RESULTS: To initiate studies of mammalian Ste20-related kinases, we identified a novel human PAK isoform, hPAK1. When expressed in yeast, hPAK1 was able to replace Ste20 in the pheromone response pathway. Chemical mutagenesis of a plasmid encoding hPAK1, followed by transformation into yeast, led to the identification of a potent constitutively active hPAK1 with a substitution of a highly conserved amino-acid residue (L107F) in the Cdc42-binding domain. Expression of the hPAK1(L107F) allele in mammalian cells led to specific activation of the Jun N-terminal kinase MAP kinase pathway, but not the mechanistically related extracellular signal-regulated MAP kinase pathway. CONCLUSIONS: These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast. Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , GTP Phosphohydrolases/metabolism , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins , Alleles , Amino Acid Sequence , Base Sequence , Cell Cycle Proteins/metabolism , Cloning, Molecular , DNA, Complementary , Enzyme Activation , GTP-Binding Proteins/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Molecular Sequence Data , Mutagenesis , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/genetics , Signal Transduction , cdc42 GTP-Binding Protein , p21-Activated KinasesABSTRACT
The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.
