Activated Cdc42/Rac reconstitutes FcɛRI-mediated Ca<sup>2+</sup>mobilization and degranulation in mutant RBL mast cells

Hong-Geller, Elizabeth; Holowka, David; Siraganian, Reuben P.; Baird, Barbara; Cerione, Richard A.

doi:10.1073/pnas.98.3.1154

Cited by 53 publications

(59 citation statements)

References 27 publications

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“…The functional defects observed in mice deficient in PLC␥ 2 are not restricted to B cells but are also observed in platelets, natural killer cells, monocytes/macrophages, and mast cells (60,61 There was no effect, however, of activated Cdc42 on PLC␥ 1 activity upon reconstitution of the two purified proteins in a cell-free system (65). At first glance, these findings are not easily consistent with the findings reported here.…”

Section: Discussioncontrasting

confidence: 55%

“…However, PLC␥ 2 was apparently not examined in the latter studies, and the results presented here do not exclude the possibility that activated Cdc42 and Rac1 interact with PLC␥ 1 and that Cdc42 interacts with PLC␥ 2 without enhancing the catalytic activities of the PLC␥ isozymes. Moreover, activated Cdc42 appeared to interact with a species of PLC␥ 1 that migrated slightly faster on SDS-polyacrylamide gels than the majority of recombinant PLC␥ 1 , suggesting that it may be a modified form of the enzyme that binds most effectively to activated Cdc42 (65).…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussioncontrasting

confidence: 55%

Section: Discussionmentioning

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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“…Patch clamp studies have established that Rac and Cdc42 regulate the early calcium release-activated calcium current (I CRAC ) as well as receptor-mediated calcium influx [23]. Expression of constitutively active mutants of Rac and Cdc42 in B6A4C1 cells, which are deficient in the calcium response, reconstituted the early (1 min) calcium influx wave [22,43]. Taken together, these observations suggest that Rac and Cdc42 are most likely upstream of PLCc.…”

Section: Discussionmentioning

confidence: 55%

“…Studies in bone marrow mast cells derived from PKCd -/-mice showed that PKCd plays a negative role in calcium mobilization and antigen-induced mast cell degranulation [46]. Given that Rac and Cdc42 activation are required for calcium signaling in mast cells [21][22][23][24]43], the effects of PKCd on degranulation could be in part via inhibition of Rac. The mechanism for PKC-mediated inhibition of Rac is yet to be determined but could involve PKC regulation of a Rac-specific guanine nucleotide exchange factor (GEF) or GAP, analogous to the inhibition of Ral-GEF by PKC [47].…”

Section: Discussionmentioning

confidence: 99%

“…Introduction of Rac into permeabilized mast cells causes exocytic secretion [19], and expression of dominant-negative forms of Rac and Cdc42 inhibits antigen-mediated degranulation [20]. This inhibition is rescued by the addition of calcium ionophores, implying that Rac and Cdc42 are upstream of the calcium influx pathway [21,22]. Patch clamp studies in the same system showed that Rac/ Cdc42 are involved in calcium mobilization, in part through the regulation of calcium release-activated calcium channels [23].…”

Section: Introductionmentioning

confidence: 99%

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Phospholipase Cγ negatively regulates Rac/Cdc42 activation in antigen‐stimulated mast cells

El‐Sibai

Backer

2006

Eur J Immunol

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The Rho GTPases Rac and Cdc42 play a central role in the regulation of secretory and cytoskeletal responses in antigen-stimulated mast cells. In this study, we examine the kinetics and mechanism of Rac and Cdc42 activation in the rat basophilic leukemia RBL-2H3 cells. The activation kinetics of both Rac and Cdc42 show a biphasic profile, consisting of an early transient peak at 1 min and a late sustained activation phase at 20-40 min. The inhibition of phospholipase C (PLC)c causes a twofold increase in Rac and Cdc42 activation that coincides with a dramatic production of atypical filopodialike structures. Inhibition of protein kinase C using bisindolylmaleimide mimics the effect of PLCc inhibition on Rac activation, but not on Cdc42 activation. In contrast, depletion of intracellular calcium leads to a complete inhibition of the early activation peak of both Rac and Cdc42, without significant effects on the late sustained activation. These data suggest that PLCc is involved in a negative feedback loop that leads to the inhibition of Rac and Cdc42. They also suggest that the presence of intracellular calcium is a prerequisite for both Rac and Cdc42 activation. IntroductionMast cells participate in the pathogenesis of immediate hypersensitivity and allergic reactions in humans [1]. Moreover, mast cells are important contributors to airway hyper-responsiveness that occurs in asthmatic inflammation [2]. In response to allergens and other exogenous stimuli, mast cells accumulate in the airway smooth muscle and in the bronchial intraepithelial layer [3]. The activation of mast cells, through the crosslinking of the FceRI, leads to the release of pre-formed early-phase inflammatory mediators and vasoactive substances from granules. It also leads to the in situ production of cytokines and bioactive lipids that contribute to the late-phase manifestation of the asthmatic reaction [4].Aggregation of FceRI on the surface of mast cells in response to multivalent ligand binding initiates a cascade of downstream signaling effectors that leads to degranulation [5]. These include phosphatidylinositol 3-kinase (PI3K), the guanine nucleotide exchange factor Vav, and phospholipase C (PLC)c1 [6]. The early activation of PLCc1 results in the generation of inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol. These two second messengers lead to increases in cytoplasmic calcium and activation of calcium/diacylglycerol-regulated isoforms of protein kinase C (PKC) which culminate in granule margination and exocytosis [7]. PI3K plays an important regulatory role as an upstream activator of PLCc1, as well as a regulator of calcium flux during mast cell degranulation [8][9][10][11][12][13].Members of the Rho family of small GTPases also regulate signaling events in mast cells [14][15][16]. In the rat basophilic leukemia (RBL-2H3) cells, Rac and Cdc42 have been implicated in the control of the FceRImediated phagocytosis and the regulation of migration, In conjunction with the secretory response, antigenstimulated mast cells undergo drama...

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Cell division cycle 42 reflects disease risk, symptoms, Th1/Th2 disproportion, and its short‐term variation indicates symptom amelioration after treatment in allergic rhinitis patients

Zhang

Xie

Fang

2022

Clinical Laboratory Analysis

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Activated Cdc42/Rac reconstitutes FcɛRI-mediated Ca²⁺mobilization and degranulation in mutant RBL mast cells

Cited by 53 publications

References 27 publications

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

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

Phospholipase Cγ negatively regulates Rac/Cdc42 activation in antigen‐stimulated mast cells

Cell division cycle 42 reflects disease risk, symptoms, Th1/Th2 disproportion, and its short‐term variation indicates symptom amelioration after treatment in allergic rhinitis patients

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Activated Cdc42/Rac reconstitutes FcɛRI-mediated Ca2+mobilization and degranulation in mutant RBL mast cells

Cited by 53 publications

References 27 publications

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

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

Phospholipase Cγ negatively regulates Rac/Cdc42 activation in antigen‐stimulated mast cells

Cell division cycle 42 reflects disease risk, symptoms, Th1/Th2 disproportion, and its short‐term variation indicates symptom amelioration after treatment in allergic rhinitis patients

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Activated Cdc42/Rac reconstitutes FcɛRI-mediated Ca²⁺mobilization and degranulation in mutant RBL mast cells