Article

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

ABSTRACT

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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    • "This positive feedback loop was first proposed by computational studies of signal transduction (Bhalla and Iyengar, 1999). While PKC-dependent activation of MAPK has been experimentally observed in many types of cells (Marais et al., 1998; Corbit et al., 2003; Zheng et al., 2005; Wen-Sheng, 2006), including Purkinje cells (Endo and Launey, 2003; Ito-Ishida et al., 2006; Tanaka and Augustine, 2008), the molecular mechanisms underlying this interaction are extremely varied and depend on cell type and stimulus conditions (Kolch et al., 1993; Marais et al., 1998; Schönwasser et al., 1998; Verin et al., 2000; Corbit et al., 2003; Zheng et al., 2005; Wen-Sheng, 2006). Thus, it remains unknown as to how PKC activates MAPK during cerebellar LTD, and therefore this critical reaction in the positive feedback loop of Purkinje cells is unidentified. "
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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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    • "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). "

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