In Vivo Receptor-Mediated Phosphorylation of a G Protein in
            <i>Dictyostelium</i>

Gundersen, Robert E.; Devreotes, Peter N.

doi:10.1126/science.2110382

Cited by 55 publications

(34 citation statements)

References 26 publications

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“…Phosphorylation of Ga2 was assessed by a shift in Ga2 mobility on SDS-PAGE as previously described [6] with the following modifications. Cells were starved at 2xl0 5 cells/ml with 50 nM pulses of cAMP delivered every 6 min.…”

Section: Ga2 Phosphorylationmentioning

confidence: 99%

“…Cells were starved at 2xl0 5 cells/ml with 50 nM pulses of cAMP delivered every 6 min. After 5 h of starvation, cells were pelleted and washed once with fresh DB and quickly resuspended in DB at three cell densities: 5X10 5 , 5X10 6 , and 5X10 7 cells/ml. Caffeine was added to the cell suspensions to 2 mM and these were shaken for 20 min at 200 rpm.…”

Section: Ga2 Phosphorylationmentioning

confidence: 99%

“…GDP is exchanged for GTP, and Ga2 becomes phosphorylated on Ser-113 [28]. Phosphorylation of Goc2 causes a characteristic mobility shift on SDS-PAGE, which can be detected by staining Western blots of whole cells with anti-Goc2 antibodies (Gundersen and Devreotes [6] and Fig. 3).…”

Section: Cell Typementioning

confidence: 99%

“…The results are the means of seven experiments and the associated standard errors of the mean. The bands above 42 kDa and below 40 kDa represent variable non-specific staining [6].…”

Section: ±5 90±4mentioning

confidence: 99%

“…The incoming cAMP pulse is sensed by cARl cell surface cAMP receptors, which cause Gcc2 subunits to release GDP and bind GTP [3][4][5]. This activation causes the G(3y subunits, along with a cytosolic protein called CRAC, to transiently activate adenylyl cyclase while the Ga2 subunits activate guanylyl cyclase and become phosphorylated [6]. Activation of cARl also causes a transient influx of Ca 2+ , which is G-protein independent (see [7][8][9][10][11] for review).…”

Section: Introductionmentioning

confidence: 99%

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A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

1997

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Starving Dictyostelium discoideum cells monitor the local density of other starving cells by simultaneously secreting and sensing CMF. CMF regulates signal transduction through the chemoattractant cAMP receptor, cARl. cARl activates a heterotrimeric G protein by stimulating Goc2 to release GDP and bind GTP. We show here that the rate of cAMP-stimulated GTP hydrolysis in membranes from cells exposed to CMF is roughly 4 times slower than in membranes from untreated cells, even though the rate of GTP binding is the same. This hydrolysis is abolished in cells lacking Goc2. Our data thus suggest that CMF regulates cAMP signal transduction in part by prolonging the lifetime of the Ga2-GTP complex.

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Section: Ga2 Phosphorylationmentioning

confidence: 99%

Section: Ga2 Phosphorylationmentioning

confidence: 99%

Section: Cell Typementioning

confidence: 99%

“…The results are the means of seven experiments and the associated standard errors of the mean. The bands above 42 kDa and below 40 kDa represent variable non-specific staining [6].…”

Section: ±5 90±4mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

1997

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Protein kinases A and C positively regulate G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha in MC3T3-E1 osteoblasts

Rapuano

Bockman

1997

J. Cell. Biochem.

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The role(s) of protein kinases in the regulation of G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha was investigated in the osteoblast cell line MC3T3-E1. We have previously reported the stimulatory effects of tumor necrosis factor-alpha and A1F4-, an activator of G proteins, on this phospholipase pathway documented by a decrease in mass of PI and release of diacylglycerol. In this study, we further explored the mechanism(s) by which the tumor necrosis factor or A1F4(-)-promoted breakdown of phosphatidylinositol and the polyphosphoinositides by phospholipase C is regulated. Tumor necrosis factor-alpha was found to elicit a 4-5-fold increase in the formation of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate; and a 36% increase in [3H]inositol-1-phosphate within 5 min in prelabeled cells. [3H]inositol-4-phosphate, a metabolite of [3H]inositol-1,4-phosphate and [3H]inositol-1,4,5-phosphate, was found to be the predominant phosphoinositol product of tumor necrosis factor-alpha and A1F4(-)-activated phospholipase C hydrolysis after 30 min. In addition, the preincubation of cells with pertussis toxin decreased the tumor necrosis factor-induced release of inositol phosphates by 53%. Inhibitors of protein kinase C, including Et-18-OMe and H-7, dramatically decreased the formation of [3H]inositol phosphates stimulated by either tumor necrosis factor-alpha or A1F4- by 90-100% but did not affect basal formation. The activation of cAMP-dependent protein kinase, or protein kinase A, by the treatment of cells with forskolin or 8-BrcAMP augmented basal, tumor necrosis factor-alpha and A1F4(-)-induced [3H]inositol phosphate formation. Therefore, we report that protein kinases can regulate tumor necrosis factor-alpha-initiated signalling at the cell surface in osteoblasts through effects on the coupling between receptor, G-protein and phosphatidylinositol-specific phospholipase C.

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Phosphorylation of the G protein α-subunit, Gα2, ofDictyostelium discoideum requires a functional and activated Gα2

Gundersen

1997

J. Cell. Biochem.

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The G alpha 2-subunit of Dictyostelium discoideum is essential to the initial stage of the cell's developmental life cycle. In response to the extracellular chemoattractant, cAMP, G alpha 2 is activated and transiently phosphorylated on serine-113 [Chen et al. (1994): J Biol Chem 269:20925-20930]. The role of G alpha 2 phosphorylation remains elusive; cells expressing the S113A, nonphosphorylated mutation of G alpha 2 appear to proceed through the developmental phase normally. To gain insight into the function of G alpha 2 phosphorylation, the conditions for G alpha 2 phosphorylation were examined using a variety of alpha-subunit point mutations and chimeras. Mutations that block the G protein activation cycle prior to or at the hydrolysis of GTP (G alpha 2-S45A, G alpha 2-G207A, and G alpha 2-Q208L) preclude G alpha 2 phosphorylation in vivo. Phosphorylation of the G alpha 2-Q208L mutation does however occur in an in vitro phosphorylation assay. It appears that G alpha 2 phosphorylation, shown previously in vivo to require the cAMP receptor, also requires signaling through the G2 pathway. Results from the in vitro assay suggest that the substrate for phosphorylation is the alpha-subunit monomer.

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In Vivo Receptor-Mediated Phosphorylation of a G Protein in Dictyostelium

Cited by 55 publications

References 26 publications

A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

A cell‐density sensing factor regulates the lifetime of a chemoattractant‐induced Gα‐GTP conformation

Protein kinases A and C positively regulate G protein-dependent activation of phosphatidylinositol-specific phospholipase C by tumor necrosis factor-alpha in MC3T3-E1 osteoblasts

Phosphorylation of the G protein α-subunit, Gα2, ofDictyostelium discoideum requires a functional and activated Gα2

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