Regulation of phospholipase C-γ1 by the actin-regulatory protein villin

2001 ◽  
Vol 281 (3) ◽  
pp. C1046-C1058 ◽  
Author(s):  
A. Panebra ◽  
S.-X. Ma ◽  
L.-W. Zhai ◽  
X.-T. Wang ◽  
S. G. Rhee ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Tyrosine Kinase ◽  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Regulatory Protein ◽  
Sh2 Domain ◽  
Dependent Manner ◽  
Kinase Gene

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-γ1(PLC-γ1) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-γ1 activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-γ1. Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-γ1. The catalytic activity of PLC-γ1was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 μM. Villin inhibited PLC-γ1 activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP2), since increasing concentrations of PIP2 reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-γ1 activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP2. In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-γ1and activates PLC-γ1 catalytic activity. Villin regulates PLC-γ1 activity by modifying its own ability to bind PIP2. This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-γ1 by villin.

scholarly journals Lyn Physically Associates With the Erythropoietin Receptor and May Play a Role in Activation of the Stat5 Pathway

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3734-3745 ◽  
Author(s):  
Hiroshi Chin ◽  
Ayako Arai ◽  
Hiroshi Wakao ◽  
Ryuichi Kamiyama ◽  
Nobuyuki Miyasaka ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Tyrosine Kinase Domain ◽  
Binding Studies ◽  
In Vitro Binding ◽  
Vitro Binding

Abstract Protein tyrosine phosphorylation plays a crucial role in signaling from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion proteins further showed that the Src homology (SH)-2 domain of Lyn specifically binds with the tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.

scholarly journals Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets

Blood ◽  
2005 ◽  
Vol 106 (2) ◽  
pp. 550-557 ◽  
Author(s):  
Swaminathan Murugappan ◽  
Haripriya Shankar ◽  
Surya Bhamidipati ◽  
Robert T. Dorsam ◽  
Jianguo Jin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Time Dependent ◽  
Dependent Manner ◽  
Src Tyrosine Kinase ◽  
Functional Implications

Abstract Thrombin has been known to cause tyrosine phosphorylation of protein kinase C δ (PKCδ) in platelets, but the molecular mechanisms and function of this tyrosine phosphorylation is not known. In this study, we investigated the signaling pathways used by protease-activated receptors (PARs) to cause tyrosine phosphorylation of PKCδ and the role of this event in platelet function. PKCδ was tyrosine phosphorylated by either PAR1 or PAR4 in a concentration- and time-dependent manner in human platelets. In particular, the tyrosine 311 residue was phosphorylated downstream of PAR receptors. Also the tyrosine phosphorylation of PKCδ did not occur in Gαq-deficient mouse platelets and was inhibited in the presence of a phospholipase C (PLC) inhibitor U73122 and calcium chelator BAPTA (5,5′-dimethyl-bis(o-aminophenoxy)ethane-N, N, N ′, N ′-tetraacetic acid), suggesting a role for Gαq pathways and calcium in this event. Both PAR1 and PAR4 caused a time-dependent activation of Src (pp60c-src) tyrosine kinase and Src tyrosine kinase inhibitors completely blocked the tyrosine phosphorylation of PKCδ. Inhibition of tyrosine phosphorylation or the kinase activity of PKCδ dramatically blocked PAR-mediated thromboxane A2 generation. We conclude that thrombin causes tyrosine phosphorylation of PKCδ in a calcium- and Src-family kinase–dependent manner in platelets, with functional implications in thromboxane A2 generation.

scholarly journals Contributions of F-BAR and SH2 Domains of Fes Protein Tyrosine Kinase for Coupling to the FcεRI Pathway in Mast Cells

2008 ◽  
Vol 29 (2) ◽  
pp. 389-401 ◽  
Author(s):  
Victor A. McPherson ◽  
Stephanie Everingham ◽  
Robert Karisch ◽  
Julie A. Smith ◽  
Christian M. Udell ◽  
...  
Keyword(s):  
Mast Cells ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Regulatory Protein ◽  
Sh2 Domain ◽  
Wild Type ◽  
Protein Tyrosine ◽  
Bar Domain ◽  
Sh2 Domains

ABSTRACT This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcεRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcεRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcεRI β chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcεRI signaling and potential regulation the actin reorganization in mast cells.

scholarly journals Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation.

1994 ◽  
Vol 14 (8) ◽  
pp. 5249-5258 ◽  
Author(s):  
C Couture ◽  
G Baier ◽  
C Oetken ◽  
S Williams ◽  
D Telford ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Sh2 Domain ◽  
Lymphoid Cells ◽  
Intact Cells ◽  
Protein Tyrosine

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

scholarly journals Lyn Physically Associates With the Erythropoietin Receptor and May Play a Role in Activation of the Stat5 Pathway

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3734-3745 ◽  
Author(s):  
Hiroshi Chin ◽  
Ayako Arai ◽  
Hiroshi Wakao ◽  
Ryuichi Kamiyama ◽  
Nobuyuki Miyasaka ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Tyrosine Kinase Domain ◽  
Binding Studies ◽  
In Vitro Binding ◽  
Vitro Binding

Protein tyrosine phosphorylation plays a crucial role in signaling from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion proteins further showed that the Src homology (SH)-2 domain of Lyn specifically binds with the tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.

scholarly journals Identification of Csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure

1997 ◽  
Vol 322 (3) ◽  
pp. 927-935 ◽  
Author(s):  
Vladimir JOUKOV ◽  
Mauno VIHINEN ◽  
Satu VAINIKKA ◽  
Janusz M. SOWADSKI ◽  
Kari ALITALO ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Inactive Form

The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vivo at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain.

Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation

1994 ◽  
Vol 14 (8) ◽  
pp. 5249-5258
Author(s):  
C Couture ◽  
G Baier ◽  
C Oetken ◽  
S Williams ◽  
D Telford ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
Sh2 Domain ◽  
Lymphoid Cells ◽  
Intact Cells ◽  
Protein Tyrosine

The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.

v-Crk-induced cell transformation: changes in focal adhesion composition and signaling

1997 ◽  
Vol 110 (3) ◽  
pp. 389-399 ◽  
Author(s):  
M.G. Nievers ◽  
R.B. Birge ◽  
H. Greulich ◽  
A.J. Verkleij ◽  
H. Hanafusa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Cell Transformation ◽  
Focal Adhesions ◽  
Subcellular Fractionation ◽  
Sh2 Domain ◽  
Focal Adhesion Proteins ◽  
Nih 3T3

v-Crk is an oncogene product in which a viral Gag sequence is fused to a cellular Crk sequence. It contains one SH2 and one SH3 domain. To gain insight into the molecular mechanisms underlying v-Crk-induced cell transformation, we studied the subcellular localization and molecular interactions of v-Crk in v-Crk-transformed NIH-3T3 cells. Our results show that v-Crk specifically localizes to focal adhesions where it induces protein tyrosine phosphorylation. Subcellular fractionation studies indicated that a significant amount of v-Crk is present in the cytoskeletal cell fraction, a fraction that includes focal adhesions. Tyrosine phosphorylated proteins, including p130CAS, were also predominantly found in the cytoskeletal fraction. We show that v-Crk induces a translocation of p130CAS to the cytoskeleton, which is accompanied by hyperphosphorylation of this protein. Mutational analyses showed that functional v-Crk SH2 domain is required for the localization of v-Crk in focal adhesions. Functional v-Crk SH2 and SH3 domains were both found to be required for the observed increase in tyrosine phosphorylation of focal adhesion proteins and for the translocation and hyperphosphorylation of p130CAS. v-Crk immunoprecipitation studies revealed that cytoskeleton-associated v-Crk interacts with both p130CAS and an unidentified tyrosine kinase. These findings suggest that formation of a focal adhesion-located complex consisting of v-Crk, a tyrosine kinase and p130CAS, which may lead to the hyperphosphorylation of p130CAS. These specific and localized signaling events may represent initial steps in the process of v-Crk-induced cell transformation.

scholarly journals JAK2 Activates TFII-I and Regulates Its Interaction with Extracellular Signal-Regulated Kinase

2001 ◽  
Vol 21 (10) ◽  
pp. 3387-3397 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Transcription Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Serum Stimulation

ABSTRACT TFII-I is a transcription factor that shuttles between the cytoplasm and nucleus and is regulated by serine and tyrosine phosphorylation. Tyrosine phosphorylation of TFII-I can be regulated in a signal-dependent manner in various cell types. In B lymphocytes, Bruton's tyrosine kinase has been identified as a TFII-I tyrosine kinase. Here we report that JAK2 can phosphorylate and regulate TFII-I in nonlymphoid cells. The activity of TFII-I on the c-fospromoter in response to serum can be abolished by dominant negative JAK2 or the specific JAK2 kinase inhibitor AG490. Consistent with this, we have also found that JAK2 is activated by serum stimulation of fibroblasts. Tyrosine 248 of TFII-I is phosphorylated in vivo upon serum stimulation or JAK2 overexpression, and mutation of tyrosine 248 to phenylalanine inhibits the ability of JAK2 to phosphorylate TFII-I in vitro. Tyrosine 248 of TFII-I is required for its interaction with and phosphorylation by ERK and its in vivo activity on the c-fos promoter. These results indicate that the interaction between TFII-I and ERK, which is essential for its activity, can be regulated by JAK2 through phosphorylation of TFII-I at tyrosine 248. Thus, like the STAT factors, TFII-I is a direct substrate of JAK2 and a signal-dependent transcription factor that integrates signals from both tyrosine kinase and mitogen-activated protein kinase pathways to regulate transcription.

scholarly journals The Endothelial Receptor Tyrosine Kinase Tie1 Activates Phosphatidylinositol 3-Kinase and Akt To Inhibit Apoptosis

2002 ◽  
Vol 22 (6) ◽  
pp. 1704-1713 ◽  
Author(s):  
Christopher D. Kontos ◽  
Eugene H. Cha ◽  
John D. York ◽  
Kevin G. Peters
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Signal Transduction Pathway ◽  
Sh2 Domain ◽  
Orphan Receptor ◽  
Dependent Manner ◽  
Genetic Studies ◽  
Akt Phosphorylation ◽  
Stimulation Of

ABSTRACT Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.

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