Mechanism of B-Cell Receptor-Induced Phosphorylation and Activation of Phospholipase C-γ2

Kim, Yeun Ju; Sekiya, Fujio; Poulin, Benoit; Bae, Yun Soo; Rhee, Sue Goo

doi:10.1128/mcb.24.22.9986-9999.2004

Cited by 116 publications

(95 citation statements)

References 54 publications

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“…These observations suggest that these proteins are phosphorylated upon pV treatment, consistent with previously published data on reversible phosphorylation of PLC␥2 (27) and clathrin heavy chain (28). The levels of PLC␥2 in the phosphoprotein fraction were reduced upon WM treatment, consistent with PLC␥2 phosphorylation being, at least partially, dependent on PI3K activation (29). Several proteins in the other molecular weight fractions were also found to be increased in IMAC eluates upon pV treatment.…”

Section: Pervanadate Induces Protein Tyr Phosphorylation and The Pi3ksupporting

confidence: 80%

“…6). Tyr-753 and Tyr-759 phosphorylation on PLC␥2 have been reported previously to be downstream of PI3K, whereas phosphorylation on Tyr-1217 appears not to depend on PI3K activity (29). We found that only Tyr-753 phosphorylation was slightly sensitive to PI3K inhibition at all time points (Fig.…”

Section: Identification Of Proteins Phosphorylated At Tyr Residues (Asupporting

confidence: 55%

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Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a nonisotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase C␥2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospho- Reversible covalent modification of proteins is a key molecular mechanism by which membrane receptor activation is converted into intracellular signaling cascades that mediate physiological change. Engagement of ligands with their receptors results in rapid and transient phosphorylation of downstream protein targets, which as a consequence modify their catalytic properties, cellular localization, and/or binding partners. As a consequence, intracellular signaling pathways can control critical aspects of metabolism, cell cycle progression, migration, differentiation, and protein synthesis. Many diseases are a direct consequence of deregulation in these processes (1, 2); these changes may be detectable by quantitative phosphoprotein analysis.The assessment of the phosphorylation status of specific proteins is commonly performed by immunochemical methods using antisera raised against a peptide containing the site of phosphorylation of the target protein. This approach is restricted to the study of known phosphorylation sites and cannot be used to monitor and discover novel phosphorylated proteins and their sites of phosphorylation. To overcome this proble...

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Section: Pervanadate Induces Protein Tyr Phosphorylation and The Pi3ksupporting

confidence: 80%

Section: Identification Of Proteins Phosphorylated At Tyr Residues (Asupporting

confidence: 55%

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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“…We and others have previously demonstrated that BTK deficiency leads to a severe mature FoB cell deficiency, despite intact T2 and mature MZ B cell subsets, termed Xid (xid) in mice (19 -22). An xid-like phenotype is also observed in animals with gene-targeted deletion of PLC-␥2, which is a direct target of BTK, and B cell linker protein (BLNK), an adaptor which facilitates BTK and PLC-␥2 interaction (23)(24)(25)(26)(27). These observations support an active role for BCR signaling, particularly the BTK/PLC-␥2 signaling axis, in the differentiation of T2 into mature FoB cells (3,4,15,25,26,28).…”

supporting

confidence: 55%

Transitional B Cell Fate Is Associated with Developmental Stage-Specific Regulation of Diacylglycerol and Calcium Signaling upon B Cell Receptor Engagement

Hoek¹,

Antony²,

Lowe³

et al. 2006

The Journal of Immunology

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Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton’s tyrosine kinase and phospholipase C-γ2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.

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“…In additional experiments (results not shown), we found that coexpression of RhoA G14V , but not of wild-type RhoA, with human PLC⑀ 1b in COS-7 cells caused an about 13.5-fold stimulation of this PLC isozyme, indicating that RhoA G14V was in fact constitutively active in this system. Activation of PLC␥ 2 by cell surface receptors has previously been shown to involve protein phosphorylation at one or several of four tyrosine residues present at positions 753, 759, 1197, and 1217 of PLC␥ 2 (47)(48)(49)(50). To examine whether phosphorylation of any one of these residues was involved in stimulation of inositol phosphate formation by activated Rac GTPases in COS-7 cells expressing PLC␥ 2 , mutants of PLC␥ 2 carrying substitutions of one, two, or all four tyrosine residues by phenylalanine residues were coexpressed with either wild-type Rac2 or Rac2 G12V .…”

Section: Resultsmentioning

confidence: 99%

Isozyme-specific Stimulation of Phospholipase C-γ2 by Rac GTPases

Piechulek

Rehlen

Walliser

et al. 2005

Journal of Biological Chemistry

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The regulation of the two isoforms of phospholipase C-␥, PLC␥ 1 and PLC␥ 2 , by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP 3 ) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLC␥ isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLC␥ may also be regulated by Rho GTPases. In this study, PLC␥ 1 and PLC␥ 2 were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLC␥ activity. PLC␥ 2 , but not PLC␥ 1 , was markedly activated in intact cells by constitutively active Rac1 G12V , Rac2 G12V , and Rac3 G12V but not by Cdc42 G12V and RhoA G14V . The mechanism of PLC␥ 2 activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLC␥ 2 -activating protein-tyrosine kinases. Activation of PLC␥ 2 by Rac2 G12V in intact cells coincided with a translocation of PLC␥ 2 from the soluble to the particulate fraction. PLC␥ isozyme-specific activation of PLC␥ 2 by Rac GTPases (Rac1 ≈ Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5-(3-O-thio)triphosphate caused a marked stimulation of PLC␥ 2 but had no effect on the activity of PLC␥ 1 . PLC␥ 1 and PLC␥ 2 have previously been shown to be indiscriminately activated by PtdInsP 3 in vitro. Thus, the results suggest a novel mechanism of PLC␥ 2 activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP 3 .Inositol phospholipid-specific phospholipases C (PLCs) 3 catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP 2 ) to produce inositol 1,4,5-trisphosphate and diacylglycerol. While the products of this reaction have long been known to act as intracellular second messengers (reviewed in Refs. 1-4), it has only recently become clear that the phospholipid substrate itself may also be considered an intracellular signaling molecule in that its local concentration in the plasma membrane and possibly in other cellular membranes regulates the activities and/or subcellular distribution of a number of regulatory or structural proteins. These include enzymes, ion channels and transporters, transcription factors, scaffolding proteins, cytoskeletal proteins, as well as proteins involved in the regulation of exocytosis, endocytosis, and membrane trafficking (reviewed in Refs. 5-8).The mammalian PLCs are divided into six subfamilies, designated ␤, ␥, ␦, ⑀, , and (reviewed in Refs. 9 and 10). The four members of the PLC␤ subfamily are activated by ␤␥ dimers of heterotrimeric G proteins and/or members of the ␣ q subfamily of G protein ␣ subunits and by certain Rho GTPases (11-14). The two members of the PLC␥ family are activated by receptor and nonreceptor protein-tyrosine kinases (reviewed in Refs. 9, 10, 15, ...

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Mechanism of B-Cell Receptor-Induced Phosphorylation and Activation of Phospholipase C-γ2

Cited by 116 publications

References 54 publications

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Transitional B Cell Fate Is Associated with Developmental Stage-Specific Regulation of Diacylglycerol and Calcium Signaling upon B Cell Receptor Engagement

Isozyme-specific Stimulation of Phospholipase C-γ2 by Rac GTPases

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