Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade.

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.

Crystal structure of the ARF-GAP domain and ankyrin repeats of PYK2-associated protein beta.

ADP ribosylation factors (ARFs), which are members of the Ras superfamily of GTP-binding proteins, are critical components of vesicular trafficking pathways in eukaryotes. Like Ras, ARFs are active in their GTP-bound form, and their duration of activity is controlled by GTPase-activating proteins (GAPs), which assist ARFs in hydrolyzing GTP to GDP. PAPbeta, a protein that binds to and is phosphorylated by the non-receptor tyrosine kinase PYK2, contains several modular signaling domains including a pleckstrin homology domain, an SH3 domain, ankyrin repeats and an ARF-GAP domain. Sequences of ARF-GAP domains show no recognizable similarity to those of other GAPs, and contain a characteristic Cys-X(2)-Cys-X(16-17)-Cys-X(2)-Cys motif. The crystal structure of the PAPbeta ARF-GAP domain and the C-terminal ankyrin repeats has been determined at 2.1 A resolution. The ARF-GAP domain comprises a central three-stranded beta-sheet flanked by five alpha-helices, with a Zn(2+) ion coordinated by the four cysteines of the cysteine-rich motif. Four ankyrin repeats are also present, the first two of which form an extensive interface with the ARF-GAP domain. An invariant arginine and several nearby hydrophobic residues are solvent exposed and are predicted to be the site of interaction with ARFs. Site-directed mutagenesis of these residues confirms their importance in ARF-GAP activity.

Identification of a new Pyk2 target protein with Arf-GAP activity.

Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal alpha-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of vesicular transport through its interaction with Pap.

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3-kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3-phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane-targeted Ras to complement a temperature-sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non-permissive temperature of 37 degreesC. Using this assay, we have identified several amino acids in the beta1-beta2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high-affinity binding to PtdIns(3, 4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain-containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non-permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

Invasion of HeLa cells by Mycoplasma penetrans and the induction of tyrosine phosphorylation of a 145-kDa host cell protein.

The ability of Mycoplasma penetrans to invade eukaryotic cells was studied using a HeLa cell line. The bactericidal antibiotic, gentamicin, in combination with low concentrations of Triton X-100, was utilized to kill mycoplasmas that had not entered the cells, allowing the quantitation of internalized organisms. The intracellular location of the mycoplasma was also documented by transmission electron microscopy. The actin polymerization inhibitor cytochalasin-D markedly inhibited the internalization process, whereas the tyrosine phosphorylation inhibitors, staurosporin and genistein had only a slight effect. As against the invasion of enteropathogenic Escherichia coli which depends on tyrosine phosphorylation of a 90-kDa (Hp90) HeLa cell protein, internalization of M. penetrans by HeLa cells was independent of the phosphorylation of Hp90. Nonetheless, tyrosine phosphorylation of a 145-kDa HeLa cell protein was found to be associated with the interaction of M. penetrans with HeLa cells.

Chemotaxis, sporulation, and larvicide production in Bacillus sphaericus 2362. The influence of L-ethionine, and of aminophenylboronic acid.

4-Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as L-ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl-accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion-related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.

Altered chemotaxis of a Bacillus sphaericus L-ethionine-resistant sporulation mutant. A probable link between chemotaxis and sporulation.

A UV irradiation-induced mutant of B. sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. The ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

Motility and chemotaxis in Bacillus sphaericus. Dependence upon stage of growth.

Chemotaxis and motility of B. sphaericus 2362 were monitored as the function of a batch culture age. It was found that both functions changed independently during growth of the culture. Motility was low until the late logarithmic stage ensued, whereafter it increased sharply. The ability of cells to respond to chemoeffectors peaked at the mid-logarithmic phase. A major methyl-accepting chemotaxis protein (P53, M(r) = 53 kDa) was identified. The extent of label incorporation in this protein from L-[methyl-3H]methionine was maximal in mid- and late logarithmic phases of the growth. Cells in stationary cultures incorporated very low amounts of the label. At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre-incubated with amino acids. Although extents of P53 labeling in mid- and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis. Supermotile sporulating cells were practically insensitive to environmental stimuli. The difference in development of sensory and locomotive functions may be interpreted as an adaptive response. A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients. Insensitive sporulating cells would tend to disperse randomly from the nutrient-exhausted area. Thus, spore formation would occur in larger volume of the habitat, increasing the chance of the microbial population to survive.

Motility and chemotaxis in Bacillus sphaericus. Dependence upon stage of growth.

Chemotaxis and motility of Bacillus sphaericus 2362 were monitored as a function of the batch culture age. It was found that both functions changed independently during growth of the culture. Motility was low until the late logarithmic stage ensued, whereafter it increased sharply. The ability of cells to respond to chemo-effectors peaked at the mid-logarithmic phase. A major methyl-accepting chemotaxis protein (P53, M(r) = 53 kDa) was identified. The extent of label incorporation in this protein from L-[methyl-3H]methionine was maximal in mid- and late-logarithmic phases of the growth. Cells in stationary cultures incorporated very low amounts of the label. At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre-incubated with amino acids. Although extents of P53 labeling in mid- and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis. Supermotile sporulating cells were practically insensitive to environmental stimuli. The difference in development of sensory and locomotive functions may be interpreted as an adaptive response. A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients. Insensitive sporulating cells would tend to disperse randomly from the nutrient-exhausted area. Thus, spore formation would occur in larger volume of the habitat, increasing the chance of microbial population to survive.

Chemotaxis, sporulation, and larvicide production in Bacillus sphaericus 2362. The influence of L-ethionine, and of aminophenylboronic acid.

4-Aminophenylboronic acid (APBA), a known inhibitor of sporulation in Bacilli, as well as L-ethionine, a known inhibitor of chemotaxis in Enterobacteria, inhibited both sporulation and chemotactic behavior but not growth of Bacillus sphaericus. Both compounds also inhibited the methyl group turnover on the methyl-accepting chemotaxis protein (P53) in this microorganism. Sporulation of B. sphaericus was inhibited only when APBA was added to the growing culture before the late logarithmic stage. It was previously demonstrated that the ability of B. sphaericus to respond to chemoattractants sharply declines at the same age of the culture. Thus, it seems plausible that the action of both inhibitors upon sporulation may be attributed to the inhibition of some regulatory pathway common to chemotaxis and sporulation and involving protein methylation. Possible exchange of the nutrient depletion-related sensory information between chemotaxis and sporulation systems at the level of methyl group transfer is discussed.

Altered chemotaxis of Bacillus sphaericus L-ethionine-resistant sporulation mutant. A probable link between chemotaxis and sporulation.

A UV irradiation-induced mutant of Bacillus sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected. The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth. Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients. Ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged. A61 also demonstrated impaired chemotaxis. In contrast to the parent strain, only few amino acids elicited chemotactic response in A61. Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude. Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B. sphaericus 2362. Their chemotaxis to amino acids was considerably improved. To some amino acids, it proved to be even stronger than in the original strain, B. sphaericus 2362. It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.