Zheng Y, Liu H, Coughlin J, Zheng J, Li L, Stone JC.. Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and RAS signaling systems in B cells. Blood 105: 3648-3654

Department of Biochemistry, University of Alberta, Edmonton AB, Canada.
Blood (Impact Factor: 10.45). 06/2005; 105(9):3648-54. DOI: 10.1182/blood-2004-10-3916
Source: PubMed


B-cell receptor (BCR) signaling activates a number of intracellular signaling molecules including phospholipase C-gamma2 (PLC-gamma2), which generates membrane diacylglycerol (DAG). DAG recruits both protein kinase C (PKC) and RasGRP family members to the membrane and contributes to their activation. We have hypothesized that membrane colocalization facilitates activation of RasGRP3 by PKC. Here we demonstrate that PKC phosphorylates RasGRP3 on Thr133 in vitro, as determined by mass spectrometry. RasGRP3 with a Thr133Ala substitution is a poor PKC substrate in vitro and a poor Ras activator in vivo. Antiphosphopeptide antibodies recognize Thr133-phosphorylated RasGRP3 in B cells after BCR stimulation or DAG analog treatment, but much less so in resting cells. PKC inhibitors block RasGRP3 Thr133 phosphorylation and Ras-extracellular signal-related kinase (Erk) signaling with a similar pattern. After stimulation of T-cell receptor (TCR) or DAG analog treatment of T cells, PKC-catalyzed phosphorylation of RasGRP1 occurs on the homologous residue, Thr184. These studies shed light on the proposed "PKC-Ras pathway" and support the hypothesis that RasGRP phosphorylation by PKC is a mechanism that integrates DAG signaling systems in T and B cells. PKC-mediated regulation of RasGRPs in lymphocytes may generate cooperative signaling in response to increases in DAG. The mast- and myeloid-selective family member RasGRP4 is regulated by different means.

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    • "We sought to define the molecular mechanism by which fMLP activates RasGRP4 . Other members of the RasGRP4 family are either argued to be activated by increased free cytosolic Ca 2 þ acting via the tandem EF - hand domain in the case of RasGRP2 ( Stefanini et al , 2009 ) ( the C1 domain of RasGRP2 does not appear to bind DAG ( Johnson et al , 2007 ) or via coincident PKC - mediated phosphorylation of T133 / T184 and binding of DAG to the C1 domain in the case of RasGRP1 / 3 ( Zheng et al , 2005 ) . RasGRP4 possesses a similar overall topology to its other family members but is very unlikely to be regulated by Ca 2 þ as its tandem EF hand - like domain lacks key residues known to be required for binding of Ca 2 þ . "
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    ABSTRACT: The molecular mechanisms by which receptors regulate the Ras Binding Domains of the PIP3-generating, class I PI3Ks remain poorly understood, despite their importance in a range of biological settings, including tumorigenesis, activation of neutrophils by pro-inflammatory mediators, chemotaxis of Dictyostelium and cell growth in Drosophila. We provide evidence that G protein-coupled receptors (GPCRs) can stimulate PLCb2/b3 and diacylglycerol- dependent activation of the RasGEF, RasGRP4 in neutrophils. The genetic loss of RasGRP4 phenocopies knock-in of a Ras-insensitive version of PI3Kc in its effects on PI3Kc-dependent PIP3 accumulation, PKB activation, chemokinesis and reactive oxygen species (ROS) formation. These results establish a new mechanism by which GPCRs can stimulate Ras, and the broadly important principle that PLCs can control activation of class I PI3Ks.
    The EMBO Journal 06/2012; 31(14):3118-29. DOI:10.1038/emboj.2012.167 · 10.43 Impact Factor
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    • "PKCs on GEFs: Members of the GRP family of RasGEFs possess C1-domains that " recognize " DAG produced by phospholipases in the membrane upon membrane receptor stimulation, and through these interactions translocate to the membranes too (Ebinu et al. 1998). The GEF activity of RasGRP1,3 is greatly enhanced by concurrent phosphorylation by DAG-activated nPKCs, at least in T-and B-cells (Roose et al., 2005; Zheng et al., 2005). PKCs may also activate SOS1, the other major RasGEF, directly by phosphorylation (Rubio et al., 2006) or indirectly, by recruiting Grb2/SOS1 complexes via the Syk tyrosine kinase (Kawakami et al., 2003). "

    Neuroblastoma - Present and Future, 02/2012; , ISBN: 978-953-307-016-2
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    • "In addition, PLCg activation also can regulate Ras and diacylglycerol regulates guanine nucleotide exchange factors and proteins, such as calcineurin, CaMKII and PKC, which act in the progression of the cell cycle (Cullen and Lockyer, 2002; Bivona et al., 2003; Zheng et al., 2005; Roderick and Cook, 2008). The irregular increase in Ca 2þ i signaling after stimulation with cytokines (Fig. 1C) is in agreement with our previous report in the primitive hematopoietic cells of bone marrow cultures (Paredes-Gamero et al., 2008). "
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    ABSTRACT: Even though the involvement of intracellular Ca(2+) Ca(i)(2+) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3) ) levels and Ca(i)(2+) release in murine and human hematopoietic stem/progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+) -dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells.
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