The nonreceptor tyrosine kinase FAK (''focal adhesion kinase'') is a key mediator of integrin signaling events controlling cellular responses to the extracellular matrix, including spreading, migration, proliferation, and survival. Integrin-ligand interactions stimulate FAK tyrosine phosphorylation and activation of FAK signaling functions. Here evidence is presented that the FAK autophosphorylation site Tyr-397 mediates a direct interaction with the C-terminal Src homology 2 domain of phospholipase C (PLC)-␥1 and that this is required for both adhesion-dependent association of the two molecules and increased inositol phosphate production in mouse embryo fibroblasts. Overexpression of FAK and PLC-␥1 in COS-7 cells increases PLC-␥1 enzymatic activity and tyrosine phosphorylation, also dependent on FAK Tyr-397. However, FAK appears incapable of directly phosphorylating PLC-␥1. These observations suggest a role for FAK in recruiting PLC-␥1 to the plasma membrane at sites of cellmatrix adhesion and there promoting its enzymatic activity, possibly by releasing the repression caused by intramolecular interactions of the PLC-␥1 Src homology domains and͞or by positioning it for phosphorylation by associated Src-family kinases. These findings expand the known signaling functions of FAK and provide mechanistic insight into integrinstimulation of PLC-␥1.

The two SH2 (Src homology domain 2) domains present in phospholipase C-␥1 (PLC-␥1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 > > pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 > > pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-␥1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-␥1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-␥1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-␥1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.A rapid cellular response to growth factor binding to cell surface receptors is the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce two second messengers: inositol 1,4,5-trisphosphate and diacylglycerol (1). Respectively, these molecules initiate the mobilization of intracellular Ca 2ϩ and activation of protein kinase C. The mechanism by which growth factors stimulate this reaction involves the tyrosine phosphorylation-dependent activation of a specific phospholipase C (PLC) isoform: PLC-␥1 or PLC-␥2 (2, 3). Other PLC isoforms, PLC 1 -␤1-4 or PLC-␦1-4, are activated by growth factor-independent or unknown mechanisms. PLC-␥1 is ubiquitously expressed and targeted disruption of its gene in mice results in embryonic lethality (4). The PLC-␥2 species has a more restricted distribution (5) and has been investigated less. A PLC-␥ gene has been identified in Drosophila and its disruption leads to aberrant eye development (6). In cell culture systems, however, the role of PLC-␥1 in the mitogenic response is unclear and has been described as both essential (7-13) and non-essential (14 -20).PLC-␥ isoforms are structurally unique within this phospholipase family as they include Src homology (SH) domains, which enable protein/protein interactions (21, 22). PLC-␥1 contains two SH2 domains, which are 35% identical in amino acid sequence, and one SH3 domain. The SH2 domains mediate the association of PLC-␥1 with autophosphorylation sites on activated receptor tyrosine kinases (23), an essential prerequisite to PLC-␥1 tyrosine phosphorylation and activation. The function of SH3 domains is to mediate association with proline-rich sequences in partner proteins; however, ...

Two approaches have been utilized to investigate the role of individual SH2 domains in growth factor activation of phospholipase C-gamma1 (PLC-gamma1). Surface plasmon resonance analysis indicates that the individual N-SH2 and C-SH2 domains are able to specifically recognize a phosphotyrosine-containing peptide corresponding to Tyr 1021 of the platelet-derived growth factor (PDGF) beta receptor. To assess SH2 function in the context of the full-length PLC-gamma1 molecule as well as within the intact cell, PLC-gamma1 SH2 domain mutants, disabled by site-directed mutagenesis of the N-SH2 and/or C-SH2 domain(s), were expressed in Plcg1(-/-) fibroblasts. Under equilibrium incubation conditions (4 degrees C, 40 min), the N-SH2 domain, but not the C-SH2 domain, was sufficient to mediate significant PLC-gamma1 association with the activated PDGF receptor and PLC-gamma1 tyrosine phosphorylation. When both SH2 domains in PLC-gamma1 were disabled, the double mutant did not associate with activated PDGF receptors and was not tyrosine phosphorylated. However, no single SH2 mutant was able to mediate growth factor activation of Ca2+ mobilization or inositol 1,4,5-trisphosphate (IP3) formation. Subsequent kinetic experiments demonstrated that each single SH2 domain mutant was significantly impaired in its capacity to mediate rapid association with activated PDGF receptors and become tyrosine phosphorylated. Hence, when assayed under physiological conditions necessary to achieve a rapid biological response (Ca2+ mobilization and IP3 formation), both SH2 domains of PLC-gamma1 are essential to growth factor responsiveness.

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Background Chronic cerebral hypoperfusion (CCH) causes white matter damage and cognitive impairment, in which astrogliosis is the major pathology. However, underlying cellular mechanisms are not well defined. Activation of Na+/H+ exchanger-1 (NHE1) in reactive astrocytes causes astrocytic hypertrophy and swelling. In this study, we examined the role of NHE1 protein in astrogliosis, white matter demyelination, and cognitive function in a murine CCH model with bilateral carotid artery stenosis (BCAS). Methods Sham, BCAS, or BCAS mice receiving vehicle or a selective NHE1 inhibitor HOE642 were monitored for changes of the regional cerebral blood flow and behavioral performance for 28 days. Ex vivo MRI-DTI was subsequently conducted to detect brain injury and demyelination. Astrogliosis and demyelination were further examined by immunofluorescence staining. Astrocytic transcriptional profiles were analyzed with bulk RNA-sequencing and RT-qPCR. Results Chronic cerebral blood flow reduction and spatial working memory deficits were detected in the BCAS mice, along with significantly reduced mean fractional anisotropy (FA) values in the corpus callosum, external capsule, and hippocampus in MRI DTI analysis. Compared with the sham control mice, the BCAS mice displayed demyelination and axonal damage and increased GFAP+ astrocytes and Iba1+ microglia. Pharmacological inhibition of NHE1 protein with its inhibitor HOE642 prevented the BCAS-induced gliosis, damage of white matter tracts and hippocampus, and significantly improved cognitive performance. Transcriptome and immunostaining analysis further revealed that NHE1 inhibition specifically attenuated pro-inflammatory pathways and NADPH oxidase activation. Conclusion Our study demonstrates that NHE1 protein is involved in astrogliosis with pro-inflammatory transformation induced by CCH, and its blockade has potentials for reducing astrogliosis, demyelination, and cognitive impairment.

Release of Ca(2+) from intracellular stores at fertilization of mammalian eggs is mediated by inositol 1,4,5-trisphosphate (IP3), but the mechanism by which the sperm initiates IP3 production is not yet understood. We tested the hypothesis that phospholipase C (PLC) activity introduced into the mouse egg as a consequence of sperm-egg fusion is responsible for causing Ca(2+) release. We demonstrated that microinjecting purified, recombinant PLCgamma1 protein into mouse eggs caused Ca(2+) oscillations like those seen at fertilization. However, the PLC activity in the minimum amount of purified PLCgamma1 protein needed to elicit Ca(2+) release when injected into eggs was approximately 500-900 times the PLC activity contained in a single sperm. This indicates that a single mouse sperm does not contain enough PLC activity to be responsible for causing Ca(2+) release at fertilization. We also examined whether phosphatidylinositol 3-kinase (PI3K) could have a role in this process, and found that several inhibitors of PI3K-mediated signaling had no effect on Ca(2+) release at fertilization.

Background‘Healthy’ aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in recent clinical trials, Relaxin-therapy for 2-days could reduce mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin’s short life-span (hours) but long-lasting protective actions led us to test the hypothesis that relaxin acts at a genomic level to reverse maladaptive remodeling in aging and HF.Methods and resultsYoung (9-month) and aged (24-month), male and female F-344/Brown Norway rats were treated with relaxin (0.4 mg/kg/day) for 2-weeks delivered by subcutaneous osmotic mini-pumps or with sodium acetate (controls). The genomic effects of aging and relaxin were evaluated by extracting RNA from the left ventricles and analyzing genomic changes by RNA-sequencing, Ingenuity Pathway Analysis, MetaCore and tissue immunohistochemistry. We found that aging promotes a native inflammatory response with distinct sex-differences and relaxin suppresses transcription of multiple genes and signaling pathways associated with inflammation and HF in both genders. In addition, aging significantly increased: macrophage infiltration and atrial natriuretic peptide levels in female ventricles, and activation of the complement cascade, whereas relaxin reversed these age-related effects.ConclusionThese data support the hypothesis that relaxin alters gene transcription and suppresses inflammatory pathways and genes associated with HF and aging. Relaxin’s suppression of inflammation and fibrosis supports its potential as a therapy for cardiovascular and inflammation-related diseases, such as HF, AF and diabetes.

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