Large-Scale Identification and Evolution Indexing of Tyrosine Phosphorylation Sites from Murine Brain

Cortactin, a predominant substrate of Src family kinases, plays an important role in Arp2/3-dependent actin polymerization in lamellipodia and membrane ruffles and was recently shown to be enriched in podosomes induced by either c-Src or phorbol ester. However, the mechanisms by which cortactin regulates podosome formation have not been determined. In this study, we showed that cortactin is required for podosome formation, using siRNA knockdown of cortactin expression in smooth muscle A7r5 cells. Treatment with phorbol ester or expression of constitutively active c-Src induced genesis of cortactin-containing podosomes as well as increase in phosphorylation of cortactin at Y421 and Y466, the Src phosphorylation sites on cortactin. The Src kinase inhibitor SU-6656 significantly inhibited formation of podosomes induced by phorbol ester and phosphorylation of cortactin, whereas PKCα inhibitor did not affect podosome formation in c-Src-transfected cells. Unexpectedly, expression of cortactin mutants containing Y421F, Y421D, Y466F, or Y466D mutated sites did not affect podosome formation or cortactin translocation to podosomes, although endogenous tyrosine-phosphorylated cortactin at Y421 and Y466 was present in podosomes. Our data indicate that 1) PKCα acts upstream of Src in phosphorylation of cortactin and podosome formation in smooth muscle cells; 2) expression of cortactin is essential for genesis of podosomes; 3) phosphorylation at Y421 and Y466 is not required for translocation of cortactin to podosomes, although phosphorylation at these sites appears to be enriched in podosomes; and 4) tyrosine phosphorylation of cortactin may be involved in regulation of stability and turnover of podosomes, rather than targeting this protein to the site of podosome formation.

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Large-scale proteomic analysis of tyrosine-phosphorylation induced by T-cell receptor or B-cell receptor activation reveals new signaling pathways

PROTEOMICS ◽

10.1002/pmic.200900011 ◽

2009 ◽

Vol 9 (13) ◽

pp. 3549-3563 ◽

Cited By ~ 39

Author(s):

Masaki Matsumoto ◽

Koji Oyamada ◽

Hidehisa Takahashi ◽

Takamichi Sato ◽

Shigetsugu Hatakeyama ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Signaling Pathways ◽

Tyrosine Phosphorylation ◽

T Cell Receptor ◽

Proteomic Analysis ◽

Large Scale ◽

Cell Receptor ◽

Receptor Activation ◽

B Cell Receptor

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Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively

Molecular and Cellular Biology ◽

10.1128/mcb.13.1.133-143.1993 ◽

1993 ◽

Vol 13 (1) ◽

pp. 133-143

Author(s):

M Valius ◽

C Bazenet ◽

A Kazlauskas

Keyword(s):

Dna Synthesis ◽

Tyrosine Phosphorylation ◽

Inositol Phosphates ◽

Platelet Derived Growth Factor ◽

Phosphorylation Sites ◽

Carboxy Terminus ◽

Mitogenic Response ◽

Stable Association ◽

64 Kda Protein ◽

Carboxy Terminal

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.

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Identification of AMPK Phosphorylation Sites Reveals a Network of Proteins Involved in Cell Invasion and Facilitates Large-Scale Substrate Prediction

Cell Metabolism ◽

10.1016/j.cmet.2015.09.009 ◽

2015 ◽

Vol 22 (5) ◽

pp. 907-921 ◽

Cited By ~ 92

Author(s):

Bethany E. Schaffer ◽

Rebecca S. Levin ◽

Nicholas T. Hertz ◽

Travis J. Maures ◽

Michael L. Schoof ◽

...

Keyword(s):

Cell Invasion ◽

Large Scale ◽

Phosphorylation Sites ◽

Ampk Phosphorylation ◽

Substrate Prediction

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Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5

International Immunology ◽

10.1093/intimm/13.2.149 ◽

2001 ◽

Vol 13 (2) ◽

pp. 149-156 ◽

Cited By ~ 11

Author(s):

Kevin M. Dennehy ◽

William F. Ferris ◽

Hanne Veenstra ◽

Linda A. Zuckerman ◽

Nigel Killeen ◽

...

Keyword(s):

T Cell ◽

Tyrosine Phosphorylation ◽

Phosphorylation Sites ◽

Transmembrane Glycoprotein

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Analysis of Serine, Threonine and Tyrosine Phosphorylation Sites with Mass Spectrometry

Techniques in Protein Chemistry IV ◽

10.1016/b978-0-12-058757-5.50024-x ◽

1993 ◽

pp. 171-178 ◽

Cited By ~ 2

Author(s):

John W. Crabb ◽

Charles Johnson ◽

Karen West ◽

Janina Buczylko ◽

Krzysztof Palczewski ◽

...

Keyword(s):

Mass Spectrometry ◽

Tyrosine Phosphorylation ◽

Phosphorylation Sites

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Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.

Molecular Biology of the Cell ◽

10.1091/mbc.4.1.49 ◽

1993 ◽

Vol 4 (1) ◽

pp. 49-57 ◽

Cited By ~ 41

Author(s):

A Kashishian ◽

J A Cooper

Keyword(s):

Growth Factor ◽

Phospholipase C ◽

Tyrosine Phosphorylation ◽

Growth Factor Receptor ◽

Mitogen Activated Protein Kinase ◽

Platelet Derived Growth Factor ◽

Phosphorylation Sites ◽

C Terminus ◽

Mitogen Activated Protein ◽

Src Homology

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.

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Large-scale phosphotyrosine proteomic profiling of rat renal collecting duct epithelium reveals predominance of proteins involved in cell polarity determination

AJP Cell Physiology ◽

10.1152/ajpcell.00300.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. C27-C45 ◽

Cited By ~ 9

Author(s):

Boyang Zhao ◽

Mark A. Knepper ◽

Chung-Lin Chou ◽

Trairak Pisitkun

Keyword(s):

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Metal Ion ◽

Large Scale ◽

Collecting Duct ◽

Affinity Purification ◽

Large Fraction ◽

Proteomic Profiling ◽

Data Set ◽

Polarity Determination

Although extensive phosphoproteomic information is available for renal epithelial cells, previous emphasis has been on phosphorylation of serines and threonines with little focus on tyrosine phosphorylation. Here we have carried out large-scale identification of phosphotyrosine sites in pervanadate-treated native inner medullary collecting ducts of rat, with a view towards identification of physiological processes in epithelial cells that are potentially regulated by tyrosine phosphorylation. The method combined antibody-based affinity purification of tyrosine phosphorylated peptides coupled with immobilized metal ion chromatography to enrich tyrosine phosphopeptides, which were identified by LC-MS/MS. A total of 418 unique tyrosine phosphorylation sites in 273 proteins were identified. A large fraction of these sites have not been previously reported on standard phosphoproteomic databases. All results are accessible via an online database: http://helixweb.nih.gov/ESBL/Database/iPY/ . Analysis of surrounding sequences revealed four overrepresented motifs: [D/E]xxY*, Y*xxP, DY*, and Y*E, where the asterisk symbol indicates the site of phosphorylation. These motifs plus contextual information, integrated using the NetworKIN tool, suggest that the protein tyrosine kinases involved include members of the insulin- and ephrin-receptor kinase families. Analysis of the gene ontology (GO) terms and KEGG pathways whose protein elements are overrepresented in our data set point to structures involved in epithelial cell-cell and cell-matrix interactions (“adherens junction,” “tight junction,” and “focal adhesion”) and to components of the actin cytoskeleton as major sites of tyrosine phosphorylation in these cells. In general, these findings mesh well with evidence that tyrosine phosphorylation plays a key role in epithelial polarity determination.

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