Annexin VII and Annexin XI Are Tyrosine Phosphorylated in Peroxovanadate-treated Dogs and in Platelet-derived Growth Factor-treated Rat Vascular Smooth Muscle Cells

The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitrophosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases. The intraperitoneal administration of peroxovanadate results in the rapid accumulation of many tyrosine-phosphorylated proteins in the liver and kidney of treated animals. The availability of large pools of tyrosine-phosphorylated proteins derived from normal tissues facilitates the purification and identification of previously unknown targets for cellular tyrosine kinases. Using this procedure, we have thus far identified four proteins in the liver and kidney of peroxovanadate-treated dogs. Two of these, annexin VII and annexin XI, were novel and had not been previously reported to be substrates of tyrosine kinases while the remaining two, ezrin and clathrin, have been reported to be tyrosine phosphorylated in some cell culture systems. In the present study, isolated proteins were identified both by sequence analysis and immunological methods. Annexin VII and annexin XI are present in cultured rat vascular smooth muscle cells and both were tyrosine phosphorylated in response to a physiological ligand, platelet-derived growth factor-BB (PDGF-BB). Furthermore, the extent of tyrosine phosphorylation in response to PDGF-BB was augmented by the co-addition of peroxovanadate to cell cultures. In vitrophosphorylation assays showed that PDGF receptor, calcium-dependent tyrosine kinase (CADTK/Pyk-2), Src kinase, and epidermal growth factor receptor all were able to phosphorylate purified annexin VII and XI on tyrosine residues. These findings confirm the usefulness of phosphatase inhibition by peroxovanadate as a tool for identifying previously unknown physiological targets for cellular protein tyrosine kinases. epidermal growth factor EGF receptor signal transducer and activator of transcription phosphate-buffered saline polyacrylamide gel electrophoresis radioimmunoprotection assay vascular smooth muscle cells platelet-derived growth factor-BB PDGF receptor Src homology 2 N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Although tyrosine-phosphorylated proteins constitute only a very small percentage of the total phosphoprotein content of cells, the phosphorylation and dephosphorylation of specific tyrosine residues plays an important regulatory role in signal transduction, cell cycle control, and differentiation (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2576) Google Scholar). Enhancement of the tyrosine phosphorylation of specific cellular proteins may be induced by treatment with appropriate cell activating ligands (growth factors, hormones, and cytokines). Many of these extracellular ligands directly or indirectly activate specific tyrosine kinases. Alternatively, we have previously reported that the simple intraperitoneal injection of a tyrosine phosphatase inhibitor (peroxovanadate) into mice, in the absence of any added ligand, results within minutes in the appearance of numerous tyrosine-phosphorylated proteins in liver and kidney. These include the EGF-R,1 insulin receptor, hepatocyte growth factor receptor, SHC, Stat 1α, Stat 1β, Stat 3, Stat 5, phospholipase Cγ, insulin receptor substrate-1, β-catenin, γ-catenin, SHP-1, SHP-2, etc., all of which were identified using antibodies to known tyrosine-phosphorylated proteins (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These results emphasize the importance of phosphatase activity on the steady-state levels of phosphotyrosine in cellular proteins and the extent to which global tyrosine kinase activity is always “on” in the intact animal (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar).In the present study, in situ administration of peroxovanadate to dog was used to generate a sufficiently large pool of tyrosine-phosphorylated proteins in liver and kidney to attempt the biochemical isolation and identification of proteins not previously known to be tyrosine phosphorylated. Among the dozens (hundreds?) of phosphotyrosine-containing proteins that could be induced in these organs of the intact animal, we now report the identification of four: annexin VII, annexin XI, clathrin heavy chain, and ezrin. Tyrosine phosphorylation of ezrin and clathrin heavy chain has been detected in some cell culture systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar). Since tyrosine phosphorylation of annexin VII and XI has not previously been reported, we present evidence for the physiological relevance of these phosphorylations: PDGF-BB induces the tyrosine phosphorylation of both annexins in rat vascular smooth muscle cells (VSMC) and both annexins are tyrosine phosphorylated in vitro by a number of receptor and cytoplasmic tyrosine kinases.DISCUSSIONIn the present study we have identified in the liver and kidney of peroxovanadate-treated dogs four tyrosine-phosphorylated proteins. Two of these proteins, annexin XI and annexin VII, have not previously been shown to be tyrosine phosphorylated in any model system and the other two, clathrin and ezrin, have not been shown to be tyrosine phosphorylated in the intact animal, although they have been detected in specialized cell systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, 19Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar).Annexin VII and XI are related members of a family of homologous proteins that bind negatively charged phospholipids in the presence of calcium. The proposed physiological functions of the annexins include phospholipase A2 inhibition, exocytosis, membrane trafficking and binding, ion channel activity, and signaling. Although the proposed functions are diverse, they are all related to the phospholipid/membrane binding properties of the are in a of tissues and cell of 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). members with of of which the for and The are annexins they in sequence and and members 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. A. Biol. Chem. 1997; Google Scholar). and annexin VII and XI are tyrosine Annexin VII and XI both have related very in and residues 1997; PubMed Scopus Google Scholar, PubMed Scopus Google only annexins previously shown to be tyrosine phosphorylated are annexin and 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Annexin was shown to be tyrosine phosphorylated in cells by the in this Cohen S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Tyrosine phosphorylation of annexin was detected in cells by Cell. 1995; Scopus Google Scholar). In phosphorylation has been reported for a number of these the physiological role of phosphorylation of annexins as of annexins is Tyrosine phosphorylation may or of the proposed physiological functions for this of VII known as has previously been shown to in with a of and with of In only the was Annexin VII has been reported to be in ion channel activity, membrane and 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. A. 1996; PubMed Scopus Google PubMed Scopus (18) Google Scholar).Annexin XI was identified in by and J. Biol. Chem. Full Text PDF PubMed Google from a and by A. J. Biol. Chem. Full Text PDF PubMed Google from as a The has been reported to be for of the protein rat A. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. T. 1995; PubMed Scopus Google Scholar, S. A. S. PubMed Scopus Google and for in J. Biol. Chem. Full Text PDF PubMed Google Scholar). A. J. Biol. Chem. Full Text PDF PubMed Google detected and not tyrosine of annexin XI in were to annexin VII or XI in kidney of and rat although both were in the liver of all and and in the kidney of dog and 5, and These not to be an to in A. K. J. Biol. Chem. Full Text PDF PubMed Google were to annexin XI in rat kidney with a The for these are other phosphotyrosine-containing proteins we clathrin and ezrin, both had previously been identified as tyrosine kinase substrates in cell systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, J. Hunter T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 19Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar, Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. D. J. Cell Biol. 1997; PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar, A. D. S. A. PubMed Scopus Google Scholar). The study the tyrosine phosphorylation of both proteins in liver and kidney of intact animals. and Brodsky A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google have that tyrosine phosphorylation of clathrin may only of clathrin as on of cell or in of the of ezrin to be for cellular by the of A. T. T. Cell Biol. 1997; PubMed Scopus Google Scholar, Bretscher A. Biol. Cell. 1995; PubMed Scopus Google Scholar, A. 1995; PubMed Scopus Google Scholar). tyrosine phosphorylation of ezrin on has been shown to be in cell by of the A. D. S. A. PubMed Scopus Google Scholar). ezrin was reported to with an on the of and by activating the in ezrin was to cells A. D. S. A. PubMed Scopus Google to annexin VII and XI as tyrosine-phosphorylated proteins the administration of peroxovanadate to dog that be a that these proteins and a that Since annexin VII and XI had not previously been reported to be tyrosine phosphorylated in any cell system and we could both proteins in rat we physiological that tyrosine phosphorylation in these rat we were able to tyrosine phosphorylation of annexin VII and XI in response to a physiological PDGF-BB Furthermore, as be peroxovanadate the response to PDGF the results with the of PDGF-BB and peroxovanadate in peroxovanadate-treated dog in which ligands are In of these in vitro kinase assays showed that Src kinase, and were able to phosphorylate the annexins and Src kinases are of the and are by PDGF T. Hunter D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar, Hunter T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, is with the and has been shown to be important for tyrosine kinase in in response to PDGF T. Hunter D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results using a of annexin VII and XI that a of phosphotyrosine was present in the annexins as was for the appearance of phosphotyrosine of with kinase as be from in 2 and are many as phosphotyrosine proteins in a of which may be the as previously identified in the (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). the number of known substrates for tyrosine is to that of the four proteins identified in had not previously been identified as tyrosine that many phosphotyrosine-containing proteins to be Although tyrosine-phosphorylated proteins constitute only a very small percentage of the total phosphoprotein content of cells, the phosphorylation and dephosphorylation of specific tyrosine residues plays an important regulatory role in signal transduction, cell cycle control, and differentiation (1Hunter T. Cell. 1995; 80: 225-236Abstract Full Text PDF PubMed Scopus (2576) Google Scholar). Enhancement of the tyrosine phosphorylation of specific cellular proteins may be induced by treatment with appropriate cell activating ligands (growth factors, hormones, and cytokines). Many of these extracellular ligands directly or indirectly activate specific tyrosine kinases. Alternatively, we have previously reported that the simple intraperitoneal injection of a tyrosine phosphatase inhibitor (peroxovanadate) into mice, in the absence of any added ligand, results within minutes in the appearance of numerous tyrosine-phosphorylated proteins in liver and kidney. These include the EGF-R,1 insulin receptor, hepatocyte growth factor receptor, SHC, Stat 1α, Stat 1β, Stat 3, Stat 5, phospholipase Cγ, insulin receptor substrate-1, β-catenin, γ-catenin, SHP-1, SHP-2, etc., all of which were identified using antibodies to known tyrosine-phosphorylated proteins (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These results emphasize the importance of phosphatase activity on the steady-state levels of phosphotyrosine in cellular proteins and the extent to which global tyrosine kinase activity is always “on” in the intact animal (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In the present study, in situ administration of peroxovanadate to dog was used to generate a sufficiently large pool of tyrosine-phosphorylated proteins in liver and kidney to attempt the biochemical isolation and identification of proteins not previously known to be tyrosine phosphorylated. Among the dozens (hundreds?) of phosphotyrosine-containing proteins that could be induced in these organs of the intact animal, we now report the identification of four: annexin VII, annexin XI, clathrin heavy chain, and ezrin. Tyrosine phosphorylation of ezrin and clathrin heavy chain has been detected in some cell culture systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar). Since tyrosine phosphorylation of annexin VII and XI has not previously been reported, we present evidence for the physiological relevance of these phosphorylations: PDGF-BB induces the tyrosine phosphorylation of both annexins in rat vascular smooth muscle cells (VSMC) and both annexins are tyrosine phosphorylated in vitro by a number of receptor and cytoplasmic tyrosine kinases. the present study we have identified in the liver and kidney of peroxovanadate-treated dogs four tyrosine-phosphorylated proteins. Two of these proteins, annexin XI and annexin VII, have not previously been shown to be tyrosine phosphorylated in any model system and the other two, clathrin and ezrin, have not been shown to be tyrosine phosphorylated in the intact animal, although they have been detected in specialized cell systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, 19Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar).Annexin VII and XI are related members of a family of homologous proteins that bind negatively charged phospholipids in the presence of calcium. The proposed physiological functions of the annexins include phospholipase A2 inhibition, exocytosis, membrane trafficking and binding, ion channel activity, and signaling. Although the proposed functions are diverse, they are all related to the phospholipid/membrane binding properties of the are in a of tissues and cell of 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). members with of of which the for and The are annexins they in sequence and and members 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. A. Biol. Chem. 1997; Google Scholar). and annexin VII and XI are tyrosine Annexin VII and XI both have related very in and residues 1997; PubMed Scopus Google Scholar, PubMed Scopus Google only annexins previously shown to be tyrosine phosphorylated are annexin and 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Annexin was shown to be tyrosine phosphorylated in cells by the in this Cohen S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Tyrosine phosphorylation of annexin was detected in cells by Cell. 1995; Scopus Google Scholar). In phosphorylation has been reported for a number of these the physiological role of phosphorylation of annexins as of annexins is Tyrosine phosphorylation may or of the proposed physiological functions for this of VII known as has previously been shown to in with a of and with of In only the was Annexin VII has been reported to be in ion channel activity, membrane and 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. A. 1996; PubMed Scopus Google PubMed Scopus (18) Google Scholar).Annexin XI was identified in by and J. Biol. Chem. Full Text PDF PubMed Google from a and by A. J. Biol. Chem. Full Text PDF PubMed Google from as a The has been reported to be for of the protein rat A. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. T. 1995; PubMed Scopus Google Scholar, S. A. S. PubMed Scopus Google and for in J. Biol. Chem. Full Text PDF PubMed Google Scholar). A. J. Biol. Chem. Full Text PDF PubMed Google detected and not tyrosine of annexin XI in were to annexin VII or XI in kidney of and rat although both were in the liver of all and and in the kidney of dog and 5, and These not to be an to in A. K. J. Biol. Chem. Full Text PDF PubMed Google were to annexin XI in rat kidney with a The for these are other phosphotyrosine-containing proteins we clathrin and ezrin, both had previously been identified as tyrosine kinase substrates in cell systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, J. Hunter T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 19Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar, Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. D. J. Cell Biol. 1997; PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar, A. D. S. A. PubMed Scopus Google Scholar). The study the tyrosine phosphorylation of both proteins in liver and kidney of intact animals. and Brodsky A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google have that tyrosine phosphorylation of clathrin may only of clathrin as on of cell or in of the of ezrin to be for cellular by the of A. T. T. Cell Biol. 1997; PubMed Scopus Google Scholar, Bretscher A. Biol. Cell. 1995; PubMed Scopus Google Scholar, A. 1995; PubMed Scopus Google Scholar). tyrosine phosphorylation of ezrin on has been shown to be in cell by of the A. D. S. A. PubMed Scopus Google Scholar). ezrin was reported to with an on the of and by activating the in ezrin was to cells A. D. S. A. PubMed Scopus Google to annexin VII and XI as tyrosine-phosphorylated proteins the administration of peroxovanadate to dog that be a that these proteins and a that Since annexin VII and XI had not previously been reported to be tyrosine phosphorylated in any cell system and we could both proteins in rat we physiological that tyrosine phosphorylation in these rat we were able to tyrosine phosphorylation of annexin VII and XI in response to a physiological PDGF-BB Furthermore, as be peroxovanadate the response to PDGF the results with the of PDGF-BB and peroxovanadate in peroxovanadate-treated dog in which ligands are In of these in vitro kinase assays showed that Src kinase, and were able to phosphorylate the annexins and Src kinases are of the and are by PDGF T. Hunter D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar, Hunter T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, is with the and has been shown to be important for tyrosine kinase in in response to PDGF T. Hunter D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results using a of annexin VII and XI that a of phosphotyrosine was present in the annexins as was for the appearance of phosphotyrosine of with kinase as be from in 2 and are many as phosphotyrosine proteins in a of which may be the as previously identified in the (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). the number of known substrates for tyrosine is to that of the four proteins identified in had not previously been identified as tyrosine that many phosphotyrosine-containing proteins to be In the present study we have identified in the liver and kidney of peroxovanadate-treated dogs four tyrosine-phosphorylated proteins. Two of these proteins, annexin XI and annexin VII, have not previously been shown to be tyrosine phosphorylated in any model system and the other two, clathrin and ezrin, have not been shown to be tyrosine phosphorylated in the intact animal, although they have been detected in specialized cell systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, 19Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar). Annexin VII and XI are related members of a family of homologous proteins that bind negatively charged phospholipids in the presence of calcium. The proposed physiological functions of the annexins include phospholipase A2 inhibition, exocytosis, membrane trafficking and binding, ion channel activity, and signaling. Although the proposed functions are diverse, they are all related to the phospholipid/membrane binding properties of the proteins. are in a of tissues and cell of 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). members with of of which the for and The are annexins they in sequence and and members 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. A. Biol. Chem. 1997; Google Scholar). and annexin VII and XI are tyrosine Annexin VII and XI both have related very in and residues 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The only annexins previously shown to be tyrosine phosphorylated are annexin and 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Annexin was shown to be tyrosine phosphorylated in cells by the in this Cohen S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Tyrosine phosphorylation of annexin was detected in cells by Cell. 1995; Scopus Google Scholar). In phosphorylation has been reported for a number of these the physiological role of phosphorylation of annexins as of annexins is Tyrosine phosphorylation may or of the proposed physiological functions for this of proteins. Annexin VII known as has previously been shown to in with a of and with of In only the was Annexin VII has been reported to be in ion channel activity, membrane and 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, S. A. 1996; PubMed Scopus Google PubMed Scopus (18) Google Scholar). Annexin XI was identified in by and J. Biol. Chem. Full Text PDF PubMed Google from a and by A. J. Biol. Chem. Full Text PDF PubMed Google from as a The has been reported to be for of the protein rat A. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. T. 1995; PubMed Scopus Google Scholar, S. A. S. PubMed Scopus Google and for in J. Biol. Chem. Full Text PDF PubMed Google Scholar). A. J. Biol. Chem. Full Text PDF PubMed Google detected and not tyrosine of annexin XI in were to annexin VII or XI in kidney of and rat although both were in the liver of all and and in the kidney of dog and 5, and These not to be an to in A. K. J. Biol. Chem. Full Text PDF PubMed Google were to annexin XI in rat kidney with a The for these are The other phosphotyrosine-containing proteins we clathrin and ezrin, both had previously been identified as tyrosine kinase substrates in cell systems (3Gould K.L. Cooper J.A. Bretscher A. Hunter T. J. Cell Biol. 1986; 102: 660-669Crossref PubMed Scopus (109) Google Scholar, 4Martin-Perez J. Bar-Zvi D. Branton D. Erikson R.L. J. Cell Biol. 1989; 109: 577-584Crossref PubMed Scopus (18) Google Scholar, 5Wilde A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google Scholar, J. Hunter T. J. Biol. Chem. Full Text PDF PubMed Google Scholar, 19Bretscher A. J. Cell Biol. 1989; 108: 921-930Crossref PubMed Scopus (334) Google Scholar, Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T. A. D. J. Cell Biol. 1997; PubMed Scopus Google Scholar, Cell PubMed Scopus Google Scholar, A. D. S. A. PubMed Scopus Google Scholar). The study the tyrosine phosphorylation of both proteins in liver and kidney of intact animals. and Brodsky A. Brodsky F.M. J. Cell Biol. 1996; 135: 635-645Crossref PubMed Scopus (132) Google have that tyrosine phosphorylation of clathrin may only of clathrin as on of cell or in of the of ezrin to be for cellular by the of A. T. T. Cell Biol. 1997; PubMed Scopus Google Scholar, Bretscher A. Biol. Cell. 1995; PubMed Scopus Google Scholar, A. 1995; PubMed Scopus Google Scholar). tyrosine phosphorylation of ezrin on has been shown to be in cell by of the A. D. S. A. PubMed Scopus Google Scholar). ezrin was reported to with an on the of and by activating the in ezrin was to cells A. D. S. A. PubMed Scopus Google Scholar). to annexin VII and XI as tyrosine-phosphorylated proteins the administration of peroxovanadate to dog that be a that these proteins and a that Since annexin VII and XI had not previously been reported to be tyrosine phosphorylated in any cell system and we could both proteins in rat we physiological that tyrosine phosphorylation in these Using rat we were able to tyrosine phosphorylation of annexin VII and XI in response to a physiological PDGF-BB Furthermore, as be peroxovanadate the response to PDGF the results with the of PDGF-BB and peroxovanadate in peroxovanadate-treated dog in which ligands are In of these in vitro kinase assays showed that Src kinase, and were able to phosphorylate the annexins and Src kinases are of the and are by PDGF T. Hunter D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. PubMed Scopus Google Scholar, Hunter T. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, is with the and has been shown to be important for tyrosine kinase in in response to PDGF T. Hunter D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results using a of annexin VII and XI that a of phosphotyrosine was present in the annexins as was for the appearance of phosphotyrosine of with kinase as be from in 2 and are many as phosphotyrosine proteins in a of which may be the as previously identified in the (2Ruff S.J. Chen K. Cohen S. J. Biol. Chem. 1997; 272: 1263-1267Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). the number of known substrates for tyrosine is to that of the four proteins identified in had not previously been identified as tyrosine that many phosphotyrosine-containing proteins to be are to T. for and rat and T. for of the cell culture and the in by for the