Alan V. Smrcka

A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits

Abstract Phosphoinositide 3 kinase (PI3K) is a key signaling enzyme implicated in receptor-stimulated mitogenesis, oxidative bursting in neutrophils, membrane ruffling, and glucose uptake. A PI3K has already been purified, cloned, and shown to be regulated by receptors that act via tyrosine kinase-dependent regulatory mechanisms. We report that an immunologically, pharmacologically, and chromatographically distinct form of PI3K activity present in neutrophils and U937 cells is specifically activated by G protein βγ subunits. This data suggests PI3Ks conform to the paradigm set by receptor regulation of phosphoinositidase Cs: different receptor transduction systems specifically regulate dedicated isoforms of effector protein.

Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible

Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene (PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCε1 expression in developing and mature glomerular podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif–containing GTPase-activating protein 1 as a new interaction partner of PLCε1. Two siblings with a missense mutation in an exon encoding the PLCε1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.

Directional sensing requires Gβγ-mediated PAK1 and PIXα-dependent activation of Cdc42

Efficient chemotaxis requires directional sensing and cell polarization. We describe a signaling mechanism involving G beta gamma, PAK-associated guanine nucleotide exchange factor (PIX alpha), Cdc42, and p21-activated kinase (PAK) 1. This pathway is utilized by chemoattractants to regulate directional sensing and directional migration of myeloid cells. Our results suggest that G beta gamma binds PAK1 and, via PAK-associated PIX alpha, activates Cdc42, which in turn activates PAK1. Thus, in this pathway, PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This G beta gamma-PAK1/PIX alpha/Cdc42 pathway is essential for the localization of F-actin formation to the leading edge, the exclusion of PTEN from the leading edge, directional sensing, and the persistent directional migration of chemotactic leukocytes. Although ligand-induced production of PIP(3) is not required for activation of this pathway, PIP(3) appears to localize the activation of Cdc42 by the pathway.

Phospholipase Cϵ: a novel Ras effector

Three classes of mammalian phosphoinositide‐specific phospholipase C (PLC) have been characterized, PLCβ, PLCγ and PLCδ, that are differentially regulated by heterotrimeric G‐proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCϵ, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C‐terminal Ras‐binding (RA) domains, RA1 and RA2. The RA2 domain binds H‐Ras in a GTP‐dependent manner, comparable with the Ras‐binding domain of Raf‐1; however, the RA1 domain binds H‐Ras with a low affinity in a GTP‐independent manner. While Gαq, Gβγ or, surprisingly, H‐Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H‐Ras stimulates PLCϵ co‐expressed in COS‐7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site‐directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras–effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.

Regulation of phospholipase C by G proteins

Specific phospholipase C enzymes can hydrolyse phosphatidylinositol 4,5-bisphosphate into two products: inositol 1,4,5-trisphosphate, which regulates the release of intracellular calcium stores, and diacylglycerol, which can stimulate protein kinase C. A new group of G proteins, the Gq subfamily, have recently been shown to mediate the regulation of this activity by a variety of hormones. How do different members of this family modulate unique phospholipase C isozymes? What is the mechanism of this regulation? How might the Gq subfamily act to modulate other important second messenger pathways? The tools to answer these questions are being rapidly developed.

Differential Targeting of Gßγ-Subunit Signaling with Small Molecules

G protein βγ subunits have potential as a target for therapeutic treatment of a number of diseases. We performed virtual docking of a small-molecule library to a site on Gβγ subunits that mediates protein interactions. We hypothesized that differential targeting of this surface could allow for selective modulation of Gβγ subunit functions. Several compounds bound to Gβγ subunits with affinities from 0.1 to 60 μM and selectively modulated functional Gβγ-protein-protein interactions in vitro, chemotactic peptide signaling pathways in HL-60 leukocytes, and opioid receptor–dependent analgesia in vivo. These data demonstrate an approach for modulation of G protein–coupled receptor signaling that may represent an important therapeutic strategy.

Small Molecule Disruption of G Protein βγ Subunit Signaling Inhibits Neutrophil Chemotaxis and Inflammation

G protein βγ subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) γ catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gβγ-dependent signaling, including Gβγ-dependent activation of PI3-kinase γ and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gβγ inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gβγ-regulation may be an effective anti-inflammation strategy.

Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.

Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)ϵ that plays a critical role in maximal β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCϵ phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCϵ-stimulated Rap1 GEF activity are both required for PLCϵ-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCϵ significantly enhances Rap activation in response to βAR stimulation in the heart. Downstream of PLCϵ hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both βAR- and Epac-stimulated increases in CICR in PLCϵ+/+ myocytes but had no effect in PLCϵ–/– myocytes. βAR and Epac activation caused membrane translocation of PKCϵ in PLCϵ+/+ but not PLCϵ–/– myocytes and small interfering RNA-mediated PKCϵ knockdown significantly inhibited both βAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited βAR- and Epac-mediated CICR in PLCϵ+/+ but not PLCϵ–/– myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and βAR-stimulated peak L-type current density are similar in PLCϵ+/+ and PLCϵ–/– myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCϵ, PKCϵ, and CamKII downstream of βAR activation.

Epac-mediated activation of phospholipase C∈ plays a critical role in β-adrenergic receptor-dependent enhancement of Ca2+ mobilization in cardiac myocytes

Recently we demonstrated that PLCϵ plays an important role in β-adrenergic receptor (βAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we have reported for the first time that a pathway downstream of βAR involving the cAMP-dependent Rap GTP exchange factor, Epac, and PLCϵ regulates CICR in cardiac myocytes. To demonstrate a role for Epac in the stimulation of CICR, cardiac myocytes were treated with an Epac-selective cAMP analog, 8-4-(chlorophenylthio)-2′-O-methyladenosine-3′,5′-monophosphate (cpTOME). cpTOME treatment increased the amplitude of electrically evoked Ca2+ transients, implicating Epac for the first time in cardiac CICR. This response is abolished in PLCϵ-/- cardiac myocytes but rescued by transduction with PLCϵ, indicating that Epac is upstream of PLCϵ. Furthermore, transduction of PLCϵ+/+ cardiac myocytes with a Rap inhibitor, RapGAP1, significantly inhibited isoproterenol-dependent CICR. Using a combination of cpTOME and PKA-selective activators and inhibitors, we have shown that βAR-dependent increases in CICR consist of two independent components mediated by PKA and the novel Epac/PLCϵ pathway. We also show that Epac/PLCϵ-dependent effects on CICR are independent of sarcoplasmic reticulum loading and Ca2+ clearance mechanisms. These data define a novel endogenous PKA-independent βAR-signaling pathway through cAMP-dependent Epac activation, Rap, and PLCϵ that enhances intracellular Ca2+ release in cardiac myocytes.

Pertussis Toxin-sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC β2 and PLC β3 through the Gβγ subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC β2 and PLC β3 were shown to be activated by the βγ subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC β3 or recombinant PLC β3 in transfected COS-7 cells. However, both receptors can clearly activate PLC β2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC β2 probably through the Gβγ subunits. These findings were further corroborated by the results that PLC β3 could only be slightly activated by Gβ1γ1 or Gβ1γ5 in the cotransfection assay, whereas the Gβγ subunits strongly activated PLC β2 under the same conditions. PLC β3 can be activated by Gαq, Gα11, and Gα16 in the cotransfection assay. In addition, the Gγ2 and Gγ3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gβγ-mediated activation of PLC β2, were able to inhibit C5a or fMLP-mediated activation of PLC β2. These Gγ mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gβγ subunits are involved in PLC β2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC β3 by Gβγ may not be the primary pathway for the receptors.