scholarly journals Functional and structural characterization of allosteric activation of phospholipase Cε by Rap1A

2020 ◽  
Vol 295 (49) ◽  
pp. 16562-16571
Author(s):  
Monita Sieng ◽  
Arielle F. Selvia ◽  
Elisabeth E. Garland-Kuntz ◽  
Jesse B. Hopkins ◽  
Isaac J. Fisher ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Small Gtpases ◽  
Ph Domain ◽  
Hydrophobic Residues ◽  
X Ray Scattering ◽  
Catalytically Active ◽  
Ef Hands ◽  
Β Adrenergic Receptors ◽  
G Protein Coupled ◽  
Phospholipase Cε

Phospholipase Cε (PLCε) is activated downstream of G protein–coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.

scholarly journals Functional and structural characterization of allosteric activation of Phospholipase Cε by Rap1A

2020 ◽  
Author(s):  
Monita Sieng ◽  
Arielle F. Selvia ◽  
Elisabeth E. Garland-Kuntz ◽  
Jesse B. Hopkins ◽  
Isaac J. Fisher ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Small Gtpases ◽  
Ph Domain ◽  
Hydrophobic Residues ◽  
X Ray Scattering ◽  
Catalytically Active ◽  
Ef Hands ◽  
Β Adrenergic Receptors ◽  
G Protein Coupled ◽  
Phospholipase Cε

ABSTRACTPhospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with small GTPases, including Rap1A and Ras. While Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability, or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of β-adrenergic receptors (β-ARs), translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation, and identified hydrophobic residues on the surface of the RA2 domain that are also necessary for activation by the GTPase. Finally, small angle X-ray scattering (SAXS) showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. This data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLC core.

scholarly journals PI3Kβ links integrin activation and PI(3,4)P2 production during invadopodial maturation

2019 ◽  
Vol 30 (18) ◽  
pp. 2367-2376 ◽  
Author(s):  
Zahra Erami ◽  
Samantha Heitz ◽  
Anne R. Bresnick ◽  
Jonathan M. Backer
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Tyrosine Kinases ◽  
Breast Cancer Cells ◽  
Matrix Metalloproteases ◽  
Ph Domain ◽  
Integrin Activation ◽  
G Protein Coupled ◽  
Insight Into

The invasion of tumor cells from the primary tumor is mediated by invadopodia, actin-rich protrusive organelles that secrete matrix metalloproteases and degrade the extracellular matrix. This coupling between protrusive activity and matrix degradation facilitates tumor invasion. We previously reported that the PI3Kβ isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and G protein–coupled receptors, is required for invasion and gelatin degradation in breast cancer cells. We have now defined the mechanism by which PI3Kβ regulates invadopodia. We find that PI3Kβ is specifically activated downstream from integrins, and is required for integrin-stimulated spreading and haptotaxis as well as integrin-stimulated invadopodia formation. Surprisingly, these integrin-stimulated and PI3Kβ-dependent responses require the production of PI(3,4)P2 by the phosphoinositide 5′-phosphatase SHIP2. Thus, integrin activation of PI3Kβ is coupled to the SHIP2-dependent production of PI(3,4)P2, which regulates the recruitment of PH domain-containing scaffolds such as lamellipodin to invadopodia. These findings provide novel mechanistic insight into the role of PI3Kβ in the regulation of invadopodia in breast cancer cells.

scholarly journals Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ

2017 ◽  
Vol 474 (23) ◽  
pp. 3903-3914 ◽  
Author(s):  
Zahra Erami ◽  
Bassem D. Khalil ◽  
Gilbert Salloum ◽  
Yanhua Yao ◽  
Jaclyn LoPiccolo ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Breast Cancer Cells ◽  
Receptor Tyrosine Kinases ◽  
Small Gtpases ◽  
G Protein Coupled Receptors ◽  
Diverse Range ◽  
Akt Activation ◽  
Direct Binding ◽  
G Protein Coupled ◽  
In Cells

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kβ, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gβγ subunits to the C2-helical domain linker of p110β, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110β. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gβγ led to an additive PI3Kβ-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kβ that cannot bind Gβγ, Gβγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kβ was blocked. Surprisingly, PI3Kβ-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110β that cannot bind Rac1. Instead of directly binding to PI3Kβ, CA-Rac1 acts by enhancing Gβγ coupling to PI3Kβ, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gβγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kβ by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gβγ coupling to PI3Kβ.

scholarly journals PI3Kβ—A Versatile Transducer for GPCR, RTK, and Small GTPase Signaling

Endocrinology ◽  
2019 ◽  
Vol 160 (3) ◽  
pp. 536-555 ◽  
Author(s):  
Anne R Bresnick ◽  
Jonathan M Backer
Keyword(s):  
G Proteins ◽  
Tyrosine Kinases ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Catalytic Subunits ◽  
Regulatory Subunits ◽  
Phosphoinositide 3 Kinase ◽  
G Protein Coupled

AbstractThe phosphoinositide 3-kinase (PI3K) family includes eight distinct catalytic subunits and seven regulatory subunits. Only two PI3Ks are directly regulated downstream from G protein–coupled receptors (GPCRs): the class I enzymes PI3Kβ and PI3Kγ. Both enzymes produce phosphatidylinositol 3,4,5-trisposphate in vivo and are regulated by both heterotrimeric G proteins and small GTPases from the Ras or Rho families. However, PI3Kβ is also regulated by direct interactions with receptor tyrosine kinases (RTKs) and their tyrosine phosphorylated substrates, and similar to the class II and III PI3Ks, it binds activated Rab5. The unusually complex regulation of PI3Kβ by small and trimeric G proteins and RTKs leads to a rich landscape of signaling responses at the cellular and organismic levels. This review focuses first on the regulation of PI3Kβ activity in vitro and in cells, and then summarizes the biology of PI3Kβ signaling in distinct tissues and in human disease.

scholarly journals The Highly Conservative Cysteine of Oncomodulin as a Feasible Redox Sensor

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 66
Author(s):  
Alisa A. Vologzhannikova ◽  
Polina A. Khorn ◽  
Marina P. Shevelyova ◽  
Alexei S. Kazakov ◽  
Victor I. Emelyanenko ◽  
...  
Keyword(s):  
Solvent Accessibility ◽  
Sensory Function ◽  
Hydrophobic Residues ◽  
Metal Free ◽  
Helix Loop Helix ◽  
Redox Sensor ◽  
Ef Hand ◽  
Functional Consequences ◽  
Ef Hands ◽  
Ph Range

Oncomodulin (Ocm), or parvalbumin β, is an 11–12 kDa Ca2+-binding protein found inside and outside of vertebrate cells, which regulates numerous processes via poorly understood mechanisms. Ocm consists of two active Ca2+-specific domains of the EF-hand type (“helix-loop-helix” motif), covered by an EF-hand domain with inactive EF-hand loop, which contains a highly conservative cysteine with unknown function. In this study, we have explored peculiarities of the microenvironment of the conservative Cys18 of recombinant rat Ocm (rWT Ocm), redox properties of this residue, and structural/functional sensitivity of rWT Ocm to the homologous C18S substitution. We have found that pKa of the Cys18 thiol lays beyond the physiological pH range. The measurement of redox dependence of rWT Ocm thiol–disulfide equilibrium (glutathione redox pair) showed that redox potential of Cys18 for the metal-free and Ca2+-loaded protein is of −168 mV and −176 mV, respectively. Therefore, the conservative thiol of rWT Ocm is prone to disulfide dimerization under physiological redox conditions. The C18S substitution drastically reduces α-helices content of the metal-free and Mg2+-bound Ocm, increases solvent accessibility of its hydrophobic residues, eliminates the cooperative thermal transition in the apo-protein, suppresses Ca2+/Mg2+ affinity of the EF site, and accelerates Ca2+ dissociation from Ocm. The distinct structural and functional consequences of the minor structural modification of Cys18 indicate its possible redox sensory function. Since some other EF-hand proteins also contain a conservative redox-sensitive cysteine located in an inactive EF-hand loop, it is reasonable to suggest that in the course of evolution, some of the EF-hands attained redox sensitivity at the expense of the loss of their Ca2+ affinity.

Pleckstrin homology domains: not just for phosphoinositides

2004 ◽  
Vol 32 (5) ◽  
pp. 707-711 ◽  
Author(s):  
M.A. Lemmon
Keyword(s):  
Subcellular Localization ◽  
Human Genome ◽  
Small Gtpases ◽  
Membrane Targeting ◽  
Ph Domain ◽  
Cellular Membranes ◽  
Pleckstrin Homology ◽  
Ph Domains ◽  
Common Domain ◽  
Homology Domains

PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.

scholarly journals ArhGAP15, a Rac-specific GTPase-activating Protein, Plays a Dual Role in Inhibiting Small GTPase Signaling

2013 ◽  
Vol 288 (29) ◽  
pp. 21117-21125 ◽  
Author(s):  
Maria Radu ◽  
Sonali J. Rawat ◽  
Alexander Beeser ◽  
Anton Iliuk ◽  
Weiguo Andy Tao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Microarray ◽  
Small Gtpases ◽  
Dual Role ◽  
Small Gtpase ◽  
Ph Domain ◽  
Gtpase Activating Protein ◽  
Mutual Inhibition ◽  
Negative Role ◽  
Pak Kinase

Signaling from small GTPases is a tightly regulated process. In this work we used a protein microarray screen to identify the Rac-specific GAP, ArhGAP15, as a substrate of the Rac effectors Pak1 and Pak2. In addition to serving as a substrate of Pak1/2, we found that ArhGAP15, via its PH domain, bound to these kinases. The association of ArhGAP15 to Pak1/2 resulted in mutual inhibition of GAP and kinase catalytic activity, respectively. Knock-down of ArhGAP15 resulted in activation of Pak1/2, both indirectly, as a result of Rac activation, and directly, as a result of disruption of the ArhGAP15/Pak complex. Our data suggest that ArhGAP15 plays a dual negative role in regulating small GTPase signaling, by acting at the level of the GTPase itself, as well interacting with its effector, Pak kinase.

scholarly journals G-protein–coupled formyl peptide receptors play a dual role in neutrophil chemotaxis and bacterial phagocytosis

2019 ◽  
Vol 30 (3) ◽  
pp. 346-356 ◽  
Author(s):  
Xi Wen ◽  
Xuehua Xu ◽  
Wenxiang Sun ◽  
Keqiang Chen ◽  
Miao Pan ◽  
...  
Keyword(s):  
G Protein ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
G Protein Coupled Receptors ◽  
Peptide Receptors ◽  
Tyrosine Kinase Signaling ◽  
Formyl Peptide Receptors ◽  
Formyl Peptide ◽  
G Protein Coupled

A dogma of innate immunity is that neutrophils use G-protein–coupled receptors (GPCRs) for chemoattractant to chase bacteria through chemotaxis and then use phagocytic receptors coupled with tyrosine kinases to destroy opsonized bacteria via phagocytosis. Our current work showed that G-protein–coupled formyl peptide receptors (FPRs) directly mediate neutrophil phagocytosis. Mouse neutrophils lacking formyl peptide receptors (Fpr1/2–/–) are defective in the phagocytosis of Escherichia coli and the chemoattractant N-formyl-Met-Leu-Phe (fMLP)-coated beads. fMLP immobilized onto the surface of a bead interacts with FPRs, which trigger a Ca2+response and induce actin polymerization to form a phagocytic cup for engulfment of the bead. This chemoattractant GPCR/Gi signaling works independently of phagocytic receptor/tyrosine kinase signaling to promote phagocytosis. Thus, in addition to phagocytic receptor-mediated phagocytosis, neutrophils also utilize the chemoattractant GPCR/Gi signaling to mediate phagocytosis to fight against invading bacteria.

scholarly journals Modulating Sphingosine-1-Phosphate receptors to improve chemotherapy delivery to Ewing sarcoma

2019 ◽  
Author(s):  
Enrica Marmonti ◽  
Hannah Savage ◽  
Aiqian Zhang ◽  
Claudia Alvarez ◽  
Miriam Morrell ◽  
...  
Keyword(s):  
G Protein ◽  
Ewing Sarcoma ◽  
Tyrosine Kinases ◽  
Tumor Vasculature ◽  
Cell Junction ◽  
Tumor Vessels ◽  
S1p Receptors ◽  
Vessel Permeability ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

ABSTRACTTumor vasculature is innately dysfunctional. Poorly functional tumor vessels inefficiently deliver chemotherapy to tumor cells; vessel hyper-permeability promotes chemotherapy delivery primarily to a tumor’s periphery. Here we identify a method for enhancing chemotherapy delivery and efficacy in Ewing sarcoma (ES) in mice by modulating tumor vessel permeability. Vessel permeability is partially controlled by the G protein-coupled Sphinosine-1-phosphate receptors 1 and 2 (S1PR1 and S1PR2) on endothelial cells. S1PR1 promotes endothelial cell junction integrity while S1PR2 destabilizes it. We hypothesize that an imbalance of S1PR1:S1PR2 is partially responsible for the dysfunctional vascular phenotype characteristic of ES and that by altering the balance in favor of S1PR1, ES vessel hyper-permeability can be reversed. In this study, we demonstrate that pharmacologic activation of S1PR1 by SEW2871 or inhibition of S1PR2 by JTE-013 caused more organized, mature, and functional tumor vessels. Importantly, S1PR1 activation or S1PR2 inhibition improved chemotherapy delivery to the tumor and anti-tumor efficacy. Our data suggests that pharmacologic targeting of S1PR1 and S1PR2 may be a useful adjuvant to standard chemotherapy for ES patients.NOVELTY AND IMPACTThis study demonstrates that Sphingosine-1-Phosphate (S1P) receptors are potential novel targets for tumor vasculature remodeling and adjuvant therapy for the treatment of Ewing Sarcoma. Unlike receptor tyrosine kinases that have already been extensively evaluated for use as vascular normalizing agents in oncology, S1P receptors are G protein-coupled receptors, which have not been well studied in tumor endothelium. Pharmacologic activators and inhibitors of S1P receptors are currently in clinical trials for treatment of auto-immune and cardiovascular diseases, indicating potential for clinical translation of this work.

Abstract 227: The Effect of PHLPP2 Removal on Cardiomyocyte Hypertrophy

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Nicole H Purcell ◽  
Courtney Moc ◽  
Giovanni Birrueta ◽  
Amy Taylor ◽  
Walter Koch ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Preliminary Data ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Cardiomyocyte Hypertrophy ◽  
Ph Domain ◽  
Akt Phosphorylation ◽  
Precise Control ◽  
Neonatal Cardiomyocytes ◽  
G Protein Coupled

Crucial cellular decisions that lead to cell growth, metabolism, proliferation, and survival are all dependent on the precise control of the phosphorylation state of proteins. The serine/threonine phosphatase, PHLPP (PH domain leucine-rich repeat protein phosphatase) has been shown to directly dephosphorylate several members of the AGC family of kinases. Knockdown of PHLPP1 by siRNA in neonatal cardiomyocytes potentiates Akt activity and phosphorylation specifically at Ser473 basally and following agonist stimulation while, the removal of PHLPP2 in cardiomyocytes does not affect Akt phosphorylation as previously reported in other cells. We hypothesize that PHLPP2 may target other AGC kinases in cardiomyocytes to regulate cardiac hypertrophy. Preliminary data suggests that removal of PHLPP2 activates fetal gene re-expression at baseline and potentiates phenylephrine (PE) induced gene expression 2 fold over siControl. Recently, G protein-coupled receptor kinase 5 (GRK5), which is an AGC kinase, has been shown to regulate cardiac hypertrophy through HDAC5 phosphorylation and de-repression of gene transcription. We wanted to determine whether PHLPP2 regulates GRK5 phosphorylation and localization in cardiomyocytes. GRK5 translocates to the nucleus following hypertrophic stimulation and we found that removal of PHLPP2 increased GRK5 translocation to the nucleus at baseline and with PE treatment compared to siControl cells. Also, removal of PHLPP2 increased nuclear export of HDAC5 at baseline and following PE treatment. Conversely, overexpression of PHLPP2 blocked nuclear translocation of GRK5 following PE treatment. Ongoing studies will determine whether PHLPP acts as a scaffold or if its phosphatase activity is necessary for inhibition of GRK5 translocation by directly measuring the phosphorylation of GRK5 in the presence and absence of PHLPP2 following hypertrophic stimulation. Our preliminary data is the first to uncover GRK5 as a novel PHLPP2 target in cardiomyocytes. Since little is known about the non-canonical regulation of GRK5, understanding whether phosphorylation and localization is regulated within the cardiomyocyte by PHLPP has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.

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