Phospholipase C-γ Contains Introns Shared by src Homology 2 Domains in Many Unrelated Proteins

Abstract Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-γ (PLC-γ) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-γ evolution and structure-function relationships within the Drosophila PLC-γ encoded by small wing (sl), we cloned and sequenced the PLC-γ homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-γ homologs in other animals. PLC-γ has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-γ SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-γ are derived from an ancestral domain that was shuffled not only into PLC-γ, but also into many other unrelated genes during animal evolution.

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Regulation of class IA PI3Ks

Biochemical Society Transactions ◽

10.1042/bst0350242 ◽

2007 ◽

Vol 35 (2) ◽

pp. 242-244 ◽

Cited By ~ 33

Author(s):

H. Wu ◽

Y. Yan ◽

J.M. Backer

Keyword(s):

Tyrosine Kinases ◽

Regulatory Subunit ◽

Sh2 Domains ◽

Src Homology 2 ◽

Pi3k Activity ◽

Wide Range ◽

Rho Family Gtpases ◽

Src Homology ◽

Rho Family

Class IA PI3Ks (phosphoinositide 3-kinases) regulate a wide range of cellular responses through the production of PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) in cellular membranes. They are activated by receptor tyrosine kinases, by Ras and Rho family GTPases, and in some cases by Gβγ subunits from trimeric G-proteins. Crystallographic studies on the related class IB PI3Kγ, and biochemical and structural studies on the class IA PI3Ks, have led to new insights into how these critical enzymes are regulated in normal cells and how mutations can lead to their constitutive activation in transformed cells. The present paper will discuss recent studies on the regulation of class I (p85/p110) PI3Ks, with a focus on the role of SH2 domains (Src homology 2 domains) in the p85 regulatory subunit in modulating PI3K activity.

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The Src Homology-2 Domains (SH2 Domains) of the Protein Tyrosine Kinase p56 lck Structure, Mechanism and Drug Design

Current Drug Targets ◽

10.2174/1389450003349074 ◽

2000 ◽

Vol 1 (4) ◽

pp. 365-386 ◽

Cited By ~ 11

Author(s):

R. Broadbridge ◽

R. Sharma

Keyword(s):

Drug Design ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Protein Tyrosine ◽

Sh2 Domains ◽

Src Homology 2 ◽

Src Homology

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PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells

AJP Cell Physiology ◽

10.1152/ajpcell.00277.2012 ◽

2013 ◽

Vol 305 (3) ◽

pp. C266-C275 ◽

Cited By ~ 5

Author(s):

Nicholas C. Zachos ◽

Luke J. Lee ◽

Olga Kovbasnjuk ◽

Xuhang Li ◽

Mark Donowitz

Keyword(s):

Sh2 Domain ◽

Signaling Proteins ◽

Multiprotein Complexes ◽

Sh2 Domains ◽

Src Homology 2 ◽

Src Inhibitor ◽

Intracellular Ca ◽

Direct Binding ◽

Src Homology

Elevated levels of intracellular Ca2+([Ca2+]i) inhibit Na+/H+exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca2+]iinhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca2+signaling proteins necessary for regulation of NHE3 activity. [Ca2+]iregulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca2+]iinhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y416phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca2+]iinhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca2+]iconditions, and 4) does not directly bind NHE3. Under elevated [Ca2+]iconditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

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Expanding the Disorder-Function Paradigm in the C-Terminal Tails of Erbbs

Biomolecules ◽

10.3390/biom11111690 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1690

Author(s):

Louise Pinet ◽

Nadine Assrir ◽

Carine van Heijenoort

Keyword(s):

Tyrosine Kinases ◽

Kinase Domain ◽

Cellular Motility ◽

Dynamic Features ◽

Sh2 Domains ◽

Intrinsically Disordered ◽

Src Homology ◽

Regulation Functions ◽

And Function

ErbBs are receptor tyrosine kinases involved not only in development, but also in a wide variety of diseases, particularly cancer. Their extracellular, transmembrane, juxtamembrane, and kinase folded domains were described extensively over the past 20 years, structurally and functionally. However, their whole C-terminal tails (CTs) following the kinase domain were only described at atomic resolution in the last 4 years. They were shown to be intrinsically disordered. The CTs are known to be tyrosine-phosphorylated when the activated homo- or hetero-dimers of ErbBs are formed. Their phosphorylation triggers interaction with phosphotyrosine binding (PTB) or Src Homology 2 (SH2) domains and activates several signaling pathways controling cellular motility, proliferation, adhesion, and apoptosis. Beyond this passive role of phosphorylated domain and site display for partners, recent structural and function studies unveiled active roles in regulation of phosphorylation and interaction: the CT regulates activity of the kinase domain; different phosphorylation states have different compaction levels, potentially modulating the succession of phosphorylation events; and prolines have an important role in structure, dynamics, and possibly regulatory interactions. Here, we review both the canonical role of the disordered CT domains of ErbBs as phosphotyrosine display domains and the recent findings that expand the known range of their regulation functions linked to specific structural and dynamic features.

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APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl

Leukemia ◽

10.1038/sj.leu.2401397 ◽

1999 ◽

Vol 13 (5) ◽

pp. 760-767 ◽

Cited By ~ 30

Author(s):

T Wakioka ◽

A Sasaki ◽

K Mitsui ◽

M Yokouchi ◽

A Inoue ◽

...

Keyword(s):

Adaptor Protein ◽

Pleckstrin Homology ◽

Sh2 Domains ◽

Src Homology 2 ◽

Src Homology ◽

Stat Pathway

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Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ

The Journal of Cell Biology ◽

10.1083/jcb.138.6.1303 ◽

1997 ◽

Vol 138 (6) ◽

pp. 1303-1311 ◽

Cited By ~ 102

Author(s):

David J. Carroll ◽

Chodavarapu S. Ramarao ◽

Lisa M. Mehlmann ◽

Serge Roche ◽

Mark Terasaki ◽

...

Keyword(s):

Fusion Protein ◽

Calcium Release ◽

Specific Inhibitor ◽

Sh2 Domain ◽

High Concentration ◽

Serotonin 2C Receptor ◽

Sh2 Domains ◽

Src Homology 2 ◽

Src Homology ◽

Domain Protein

Although inositol trisphosphate (IP3) functions in releasing Ca2+ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP3. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca2+ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca2+ release initiated by the serotonin 2c receptor, or activation of Ca2+ release by IP3 injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca2+ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP3 that causes intracellular Ca2+ release.

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Anemia, thrombocytopenia, leukocytosis, extramedullary hematopoiesis, and impaired progenitor function in Pten+/-SHIP-/- mice: a novel model of myelodysplasia

Blood ◽

10.1182/blood-2003-09-3262 ◽

2004 ◽

Vol 103 (12) ◽

pp. 4503-4510 ◽

Cited By ~ 23

Author(s):

Jennifer L. Moody ◽

Lixin Xu ◽

Cheryl D. Helgason ◽

Frank R. Jirik

Keyword(s):

Extramedullary Hematopoiesis ◽

Pi3k Pathway ◽

Phosphatidylinositol 3 Kinase ◽

Short Term ◽

Src Homology 2 ◽

Donor Cells ◽

Src Homology ◽

Novel Model ◽

Pheno Type

Abstract The myeloproliferative disorder of mice lacking the Src homology 2 (SH2)-containing 5′ phosphoinositol phosphatase, SHIP, underscores the need for closely regulating phosphatidylinositol 3-kinase (PI3K) pathway activity, and hence levels of phosphatidylinositol species during hematopoiesis. The role of the 3′ phosphoinositol phosphatase Pten in this process is less clear, as its absence leads to embryonic lethality. Despite Pten heterozygosity being associated with a lymphoproliferative disorder, we found no evidence of a hematopoietic defect in Pten+/- mice. Since SHIP shares the same substrate (PIP3) with Pten, we hypothesized that the former might compensate for Pten haploinsufficiency in the marrow. Thus, we examined the effect of Pten heterozygosity in SHIP-/- mice, predicting that further dysregulation of PIP3 metabolism would exacerbate the pheno-type of the latter. Indeed, compared with SHIP-/- mice, Pten+/-SHIP-/- animals developed a myelodysplastic phenotype characterized by increased hepatosplenomegaly, extramedullary hematopoiesis, anemia, and thrombocytopenia. Consistent with a marrow defect, clonogenic assays demonstrated reductions in committed myeloid and megakaryocytic progenitors in these animals. Providing further evidence of a Pten+/-SHIP-/- progenitor abnormality, reconstitution of irradiated mice with marrows from these mice led to a marked defect in short-term repopulation of peripheral blood by donor cells. These studies suggest that the regulation of the levels and/or ratios of PI3K-derived phosphoinositol species by these 2 phosphatases is critical to normal hematopoiesis. (Blood. 2004;103:4503-4510)

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Phosphatidylinositol 3,4,5-Trisphosphate Directs Association of Src Homology 2-containing Signaling Proteins with Gelsolin

Journal of Biological Chemistry ◽

10.1074/jbc.m107494200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47434-47444 ◽

Cited By ~ 70

Author(s):

Meenakshi A. Chellaiah ◽

Rajat S. Biswas ◽

David Yuen ◽

Ulises M. Alvarez ◽

Keith A. Hruska

Keyword(s):

Bone Resorption ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Protein Interactions ◽

Lipid Extraction ◽

Focal Adhesions ◽

Amino Acid Sequences ◽

Signaling Proteins ◽

Src Homology 2 ◽

Src Homology

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130Cas, focal adhesion kinase, integrin αvβ3, vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of αvβ3-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.

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