Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the ␣ subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein ␥ subunit-like (GGL) domain between its Dishevelled͞Egl-10͞Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different G ␤ subunits reveals specific interaction between RGS11 and G ␤5 . The expression of mRNA for RGS11 and G ␤5 in human tissues overlaps. The G ␤5 ͞RGS11 heterodimer acts as a GAP on G ␣o , apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for G ␣ proteins and form complexes with specific G ␤ subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.Proteins belonging to the RGS (regulators of G protein signaling) family constitute a newly appreciated group of at least 20 mammalian gene products that act as GTPaseactivating proteins (GAPs) on the ␣ subunits of heterotrimeric, signal-transducing G proteins (1-3). As such, RGS proteins can serve as negative regulators of G proteinmediated signaling pathways by speeding the inactivation of GTP-bound G ␣ subunits. Although several members of the RGS family are relatively simple Ϸ25 kDa proteins that contain little more than a characteristic RGS domain, others include modules that impart additional functions. For example, RGS12 can associate in vitro with certain G protein-coupled receptors by virtue of an alternatively spliced PDZ (PSD-95͞ Dlg͞Z0-1) domain (4), and p115, a guanine nucleotide exchange factor for the low-molecular-weight GTPase rho, contains an RGS domain that imparts sensitivity to regulation by G protein ␣ subunits (5, 6).We describe here a novel G protein ␥ subunit-like domain (GGL; pronounced giggle) that is found in several mammalian RGS proteins (RGS6, RGS7, RGS9, and RGS11) and in EGL-10, an RGS protein of Caenorhabditis elegans. The GGL domains of RGS11 and RGS7 interact preferentially with the G protein ␤ 5 subunit, and the complex of RGS11 and ␤ 5 has GAP activity toward the G protein ␣ o subunit. MATERIALS AND METHODSGeneration of Expression Constructs. cDNAs for RGS11 and various G protein subunits were cloned from human brain or retinal mRNA, from mouse retinal mRNA, or were obtained as described (7,8); all amplified cDNAs were verified by sequencing. Human RGS7 cDNA was a kind gift of Paul F. Worley (Johns Hopkins University). cDNAs encoding G protein subunits were subcloned into the mammalian expression vector pcDNA3.1-Zeo (Invitrogen), and G ␥ and RGS protein cDNAs were subcloned in-frame with an N-terminal tandem hemagglutinin (HA)-epitope tag into a modified pcDNA3.1 vector. Recombinant baculoviruses expressing native or hexahistidine-tagged RGS11 or G ␤5 subunits were generated by using the Bac-To-Bac system by following the manufacturer's protocols (Life Technologies, Gaithersburg, MD).In Vitro Transcription and Translation. Reactions were performed using the T...

The heavy chain of myosin-ID isolated from Dictyostelium was identified as an in vitro substrate for members of the Ste20p family of serine/threonine protein kinases which are thought to regulate conserved mitogen-activated protein kinase pathways. Yeast Ste20p and Cla4p and mammalian p21-activated protein kinase (PAK) phosphorylated the heavy chain to 0.5-0.6 mol of P i /mol and stimulated the actin-dependent Mg 2؉ -ATPase activity to an extent equivalent to that of the Ste20p-like myosin-I heavy chain kinase isolated from Dictyostelium. PAK purified from rat brain required GTP␥S-Cdc42 to express full activity, whereas recombinant mouse mPAK3 fused to glutathione S-transferase and purified from bacteria, and Ste20p and Cla4p purified from yeast extracts were fully active without GTP␥S-Cdc42. These results suggest, together with the high degree of structural and functional conservation of Ste20p family members and myosin-I isoforms, that myosin-I activation by Ste20p family protein kinases may contribute to the regulation of morphogenetic processes in organisms ranging from yeast to mammalian cells.

We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS-gel electrophoresis that can stimulate the actin-activated MgATPase activity of Dictyostelium myosin ID (MyoD). In the presence of MgATP the 110-kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of MyoD. Phosphorylation to 0.5 mol of Pi/mol increased the MyoD actin-activated MgATPase rate from 0.2 to 3 mumol/min/mg. Renaturation following SDS-gel electrophoresis demonstrated that the 110-kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110-kDa protein kinase phosphorylated MyoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110-kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9-11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (MyoB) and MyoD were poor substrates for the 110-kDa protein kinase. The 110-kDa protein kinase was unable to phosphorylate the MyoB isozyme suggesting that it may be specific for MyoD.

Dictyostelium discoideum

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...

Dictyostelium discoideum MyoD, a long-tailed class I myosin, co-purified with two copies of a 16 kDa light chain. Sequence analysis of the MyoD light chain showed it to be a unique protein, termed MlcD, that shares 44% sequence identity with Dictyostelium calmodulin and 43% sequence identity with Acanthamoeba castellanii myosin IC light chain. MlcD comprises four EF-hands; however, EF-hands 2-4 contain mutations in key Ca2+-co-ordinating residues that would be predicted to impair Ca2+ binding. Electrospray ionization MS of MlcD in the presence of Ca2+ and La3+ showed the presence of one major and one minor metal-binding site. MlcD contains a single tryptophan residue (Trp39), the fluorescence intensity of which was quenched upon addition of Ca2+ or Mg2+, yielding apparent dissociation constants ( K'(d)) of 52 microM for Ca2+ and 450 microM for Mg2+. The low affinity of MlcD for Ca2+ indicates that it cannot function as a sensor of physiological Ca2+. Ca2+ did not affect the binding of MlcD to MyoD or to either of the two MyoD IQ (Ile-Gln) motifs. FLAG-MlcD expressed in Dictyostelium formed a complex with MyoD, but not with the two other long-tailed Dictyostelium myosin I isoenzymes, MyoB and MyoC. Through its specific association with the Ca2+-insensitive MlcD, MyoD may exhibit distinct regulatory properties that distinguish it from myosin I isoenzymes with calmodulin light chains.

The Dictyostelium myosin I heavy chain kinase (MI-HCK) is a member of the p21-activated kinase family (Lee, S.-F., Egelhoff, T. T., Mahasneh, A., and Cô té , G. P.

No abstract