Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

Yaciuk, P; Choi, J K; Shalloway, David

doi:10.1128/mcb.9.6.2453

Cited by 22 publications

(11 citation statements)

References 26 publications

(53 reference statements)

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“…Hirota et al reported that overexpressed c-src mutated at this residue exhibits a slight elevation in kinase activity in chicken embryo fibroblasts with no morphological alteration (23). Similarly, mutation of pp60c-sr and pp6Oc-src(F527) at Ser-17 does not affect the specific activity or transforming activity of these proteins in NIH 3T3 cells (57). We found that overexpression of c-src mutated at Ser-17 in 10T1/2 fibroblasts was not associated with a striking alteration in either specific kinase activity of the protein or agonist-induced cAMP accumulation, indicating that phosphorylation on this residue may not contribute to potentiation of 13-agonist signalling mediated by c-src kinase.…”

Section: Discussionmentioning

confidence: 99%

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Moyers¹,

Bouton²,

Parsons³

1993

Mol. Cell. Biol.

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Previously we demonstrated that C3H1OT½ murine fibroblasts overexpressing avian c-src exhibit elevated levels of cyclic AMP (cAMP) in response to 13-adrenergic believed to be important for signalling processes. In this study we show that the sites of phosphorylation by protein kinase C (PKC) (Ser-12 and Ser-48) as well as the SH2 region of pp60C are required for the enhanced response of c-src overexpressors to 13-agonist stimulation. Mutation at the site of myristylation (Gly-2) results in a decrease in the enhanced response, while mutation at the site of phosphorylation by cAMP-dependent protein kinase (Ser-17) has no effect. Two-dimensional phosphotryptic analyses indicate that phosphorylation on Ser-12 and Ser-48 in unstimulated cells is associated with the ability of overexpressed pp60csrc to potentiate 13-adrenergic signalling. Cells overexpressing wild-type c-src also exhibit enhanced cAMP accumulation upon treatment with cholera toxin, an effect that is abated in cells overexpressing pp60`c

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Section: Discussionmentioning

confidence: 99%

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Moyers¹,

Bouton²,

Parsons³

1993

Mol. Cell. Biol.

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“…4A): spot 1 (protein kinase A-phosphorylated phosphoserine 17 [27]), spots 3 and 4 (PTyr-527 [7]), spots 5 and 6 (protein kinase C-phosphorylated phosphoserine 12 [14,43)], and spot 7 (protein kinase C-phosphorylated phosphoserine 48 [14,43]). c-Src(LN), c-Src(R295), and c-Src(R295/LN) from unsynchronized cells displayed similar maps except that spots 5, 6, and 7 (Ser-12 and Ser-48) were not observed in the nonmyristylated mutants ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

Bagrodia¹,

Taylor²,

Shalloway³

1993

Mol. Cell. Biol.

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The chicken proto-oncoprotein c-Src is phosphorylated by p34cdc2 during mitosis concomitant with increased c-Src tyrosine kinase activity. On the basis of indirect evidence, we previously suggested that this is caused by partial dephosphorylation at Tyr-527, the phosphorylation of which suppresses c-Src kinase activity. In support of this hypothesis, we now show that treatment of cells with a protein tyrosine phosphatase inhibitor, sodium vanadate, blocks the mitotic increase in Src kinase activity. Also, we show that an amino-terminal mutation that prevents myristylation (and membrane localization) of c-Src does not interfere with the p34cdc2-mediated phosphorylations but blocks both mitotic dephosphorylation of Tyr-527 (in kinase-defective Src) and stimulation of c-Src kinase activity. Furthermore, in unsynchronized cells, the kinase activity of nonmyristylated c-Src is suppressed by 60% relative to wild-type c-Src, presumably because of increased Tyr-527 phosphorylation. Consistent with this, the Tyr-527 dephosphorylation rate measured in cell homogenates is much higher for wild-type, myristylated c-Src than for nonmyristylated c-Src. Tyr-527 phosphatase activity was primarily associated with the nonsoluble subcellular fraction. These findings suggest that the phosphatase(s) that acts on Tyr-527 is membrane bound and indicate that membrane localization of c-Src is necessary for its mitotic activation by dephosphorylation of Tyr-527.Chicken c-src is the cellular homolog of the Rous sarcoma virus transforming gene v-src. Both genes encode membrane-associated 60-kDa phosphoproteins with tyrosine kinase activity (25). Association of v-Src and c-Src with cellular membranes requires covalent attachment of myristate to Gly at position 2, and mutation of this residue prevents myristylation and membrane binding (3, 12,31,33,34). Other amino-terminal sequence determinants are also involved in membrane association (18), and at least for v-Src, association may involve a 32-kDa Src receptor protein (29, 30). Myristylation is not required for the mitogenic effect of v-Src (4) but is required for transformation of chicken embryo fibroblasts by v-Src (10, 17) and activated c-Src (31, 41) (although our preliminary results suggest that nonmyristylated, activated c-Src is weakly transforming in mouse fibroblasts).During mitosis, c-Src is phosphorylated by p34cdc2 or a related kinase at three amino-terminal serine and threonine residues, and c-Src kinase activity is increased (6,22,35,38,40). These mitosis-specific phosphorylations (MSPs) retard c-Src electrophoretic mobility but, by themselves, do not stimulate its kinase activity (22, 38). C-Src kinase activity can be negatively regulated by phosphorylation of its C-terminal Tyr-527 residue (5, 20, 28), and we (1, 37) and others (16) have proposed that a 10 to 20% decrease in Tyr-527 phosphorylation is the direct cause of its mitotic activation. Although we cannot reliably directly detect such a small decrease, the kinase activities of c-Src mutants lacking Tyr-527 are not fu...

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“…Alternatively there is evidence that c-Src is phosphorylated by PKA at serine 17 (38,39), and this phosphorylation is known to decrease the hydrophobicity of c-Src and enhance the release of c-Src from the cytoplasmic membrane to the cytosol (21,40,41). PKA may therefore increase translocation of c-Src from the cell membrane to the cytosol, thus decreasing its ability to phosphorylate a relevant substrate and modulate NMDA channels.…”

Section: Role Of C-src In the Pdgf-induced Depression-intracellularmentioning

confidence: 99%

Platelet-derived Growth Factor Receptor-induced Feed-forward Inhibition of Excitatory Transmission between Hippocampal Pyramidal Neurons

Lei¹,

Xiong³

et al. 1999

Journal of Biological Chemistry

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Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity of N-methyl-D-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-␥, and by blocking the intracellular release of Ca 2؉ . Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca 2؉ -and PKA-dependent inhibition of their modulation by c-Src.

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Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A.

Cited by 22 publications

References 26 publications

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

The sites of phosphorylation by protein kinase C and an intact SH2 domain are required for the enhanced response to beta-adrenergic agonists in cells overexpressing c-src.

Myristylation is required for Tyr-527 dephosphorylation and activation of pp60c-src in mitosis.

Platelet-derived Growth Factor Receptor-induced Feed-forward Inhibition of Excitatory Transmission between Hippocampal Pyramidal Neurons

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