Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs.
View Article and Find Full Text PDFTreatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in , most frequently resulting in the C481S substitution, and disclosed many mutations in , encoding phospholipase C-γ (PLCγ). The PLCγ variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases pleen-associated trosine inase (SYK) and ck/es-related ovel tyrosine kinase (LYN).
View Article and Find Full Text PDFDepending on its occurrence in the germline or somatic context, a single point mutation, S707Y, of phospholipase C-γ (PLCγ) gives rise to two distinct human disease states: acquired resistance of chronic lymphocytic leukemia cells (CLL) to inhibitors of Brutons´s tyrosine kinase (Btk) and dominantly inherited autoinflammation and PLCγ-associated antibody deficiency and immune dysregulation, APLAID, respectively. The functional relationships of the PLCγS707Y mutation to other mutations causing (i) Btk inhibitor resistance of CLL cells and (ii) the APLAID-related human disease PLCγ-associated antibody deficiency and immune dysregulation, PLAID, revealing different clinical characteristics including cold-induced urticaria, respectively, are currently incompletely understood. Here, we show that PLCγS707 point mutants displayed much higher activities at 37° C than the CLL Btk inhibitor resistance mutants R665W and L845F and the two PLAID mutants, PLCγΔ19 and PLCγΔ20-22.
View Article and Find Full Text PDFMutations in the gene encoding phospholipase C-γ (PLCγ) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ an ∼2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells.
View Article and Find Full Text PDFDeletions in the gene encoding signal-transducing inositol phospholipid-specific phospholipase C-γ2 (PLCγ2) are associated with the novel human hereditary disease PLAID (PLCγ2-associated antibody deficiency and immune dysregulation). PLAID is characterized by a rather puzzling concurrence of augmented and diminished functions of the immune system, such as cold urticaria triggered by only minimal decreases in temperature, autoimmunity, and immunodeficiency. Understanding of the functional effects of the genomic alterations at the level of the affected enzyme, PLCγ2, is currently lacking.
View Article and Find Full Text PDFThe Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction.
View Article and Find Full Text PDFIn this issue of Structure, Bunney and colleagues use a combination of NMR, SAXS, crystallography, ITC, and biochemical methods to elucidate, in molecular detail, the sequence of events causing receptor-mediated activation of phospholipase C-γ(1) by protein tyrosine phosphorylation.
View Article and Find Full Text PDFThe human cytomegalovirus (HCMV) UL78 ORF is considered to encode an orphan 7-transmembrane receptor. However, until now, the UL78 protein (pUL78) has not been characterized. Here, we have investigated the expression of pUL78 and found it mainly associated with the endoplasmic reticulum.
View Article and Find Full Text PDFWe performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells.
View Article and Find Full Text PDFWe combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-beta(2) (PLCbeta(2)) by G protein alpha(q) and betagamma dimers. We show that activation by alpha(q) and betagamma differ from activation by Rac2 and from each other. Stimulation by alpha(q) enhanced the plasma membrane association of PLCbeta(2), but not of PLCbeta(2)Delta, which lacks the alpha(q)-interacting region.
View Article and Find Full Text PDFRho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)).
View Article and Find Full Text PDFSeveral isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation.
View Article and Find Full Text PDFThe regulation of the two isoforms of phospholipase C-gamma, PLCgamma(1) and PLCgamma(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 PLCgamma isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCgamma may also be regulated by Rho GTPases.
View Article and Find Full Text PDFPhospholipase C-beta (PLCbeta) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta(2) isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42).
View Article and Find Full Text PDFPhospholipase C-beta(2) (PLC beta(2)) is activated both by heterotrimeric G protein alpha- and beta gamma- subunits and by Rho GTPases. In this study, activated Rho GTPases are shown to stimulate PLC beta isozymes with the rank order of PLC beta(2) > PLC beta(3) > or = PLC beta(1). The sensitivity of PLC beta isozymes to Rho GTPases was clearly different from that observed for G protein beta gamma dimers, which decreased in the following order: PLC beta(3) > PLC beta(2) > PLC beta(1) for beta(1)gamma(1/2) and PLC beta(2) > PLC beta(1) >>> PLC beta(3) for beta(5)gamma(2).
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