Cellular Distribution and Functions of Wild-Type and Constitutively ActivatedDictyosteliumPakB

Abstract

Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis. INTRODUCTIONMembers of the p21-activated kinase (PAK) family are key regulators of the actin cytoskeleton and cell motility in organisms ranging from yeast to mammals (Bokoch, 2003). PAKs are characterized by the presence of two conserved domains: a p21-binding domain (PBD) and a C-terminal Ser/Thr protein kinase catalytic domain. The PBD mediates interactions with active Cdc42 and Rac GTPases and encompasses an autoinhibitory sequence that potently suppresses the activity of the catalytic domain. The binding of GTPCdc42/Rac to the PBD disrupts the autoinhibitory interaction, permitting a series of autophosphorylation events that maximize kinase activity.Studies on Dictyostelium discoideum have provided valuable insights into the signaling pathways that regulate cell polarization and chemotaxis (Merlot and Firtel, 2003). To date, three Dictyostelium PAKs have been identified: PakA (Chung and Firtel, 1999), PakB (Lee et al., 1996), and PakC (GenBank accession no. AF277804). The three PAK isoforms share between 50 and 70% sequence identity within the PBD and catalytic domains but exhibit no homology outside of these regions. Loss of PakA produces cytokinesis defects in cells grown in suspension culture and prevents cells from suppressing lateral pseudopod extension or from properly retracting the cell posterior during chemotaxis (Chung and Firtel, 1999;Chung et al., 2001). These authors conclude that PakA, which localizes to the posterior of migrating cells, functions to promote the assembly of myosin II into bipolar filaments. In a second study, PakA-null cells were found to display no detectable defects with respect to locomotion or cytokinesis (Muller-Taubenberger et al., 2002).PakB was initially identified through its ability to phosphorylate and activate MyoD, a single-headed type I myosin (Lee and Côté, 1995;Lee et al., 1996). PakB was originally called myosin I heavy cha...

Role of RacC for the Regulation of WASP and Phosphatidylinositol 3-Kinase during Chemotaxis of Dictyostelium

“…Yeast Two-hybrid Assays-Yeast two-hybrid assays were performed as described previously (18). The protocols of the Matchmaker Two-hybrid system from Clontech were followed for all experiments dealing with two-hybrid assays.…”

RacG Regulates Morphology, Phagocytosis, and Chemotaxis

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Chemotactic cell movement during Dictyostelium development and gastrulation

“…PAKb appears to be involved in the regulation of myosin I function at the leading edge [30]. PAKc is involved in the inhibition of lateral pseudopods and acts through an unknown mechanism [31].…”