Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation

The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner. The mechanisms involved in the mechanical loading-induced increase in bone formation remain unclear. In this study, we showed that cyclic strain (CS) (10 min, 1% stretch at 0.25 Hz) stimulated the proliferation of overnight serum-starved ROS 17/2.8 osteoblast-like cells plated on type I collagen-coated silicone membranes. This increase was blocked by MEK inhibitor PD-98059. Signaling events were then assessed 0 min, 30 min, and 4 h after one CS period with Western blotting and coimmunoprecipitation. CS rapidly and time-dependently promoted phosphorylation of both ERK2 at Tyr-187 and focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, leading to the activation of the Ras/Raf/MEK pathway. Cell transfection with FAK mutated at Tyr-397 completely blocked ERK2 Tyr-187 phosphorylation. Quantitative immunofluorescence analysis of phosphotyrosine residues showed an increase in focal adhesion plaque number and size in strained cells. CS also induced both Src-Tyr-418 phosphorylation and Src to FAK association. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 did not prevent CS-induced FAK-Tyr-397 phosphorylation suggesting a Src-independent activation of FAK. CS also activated proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase highly homologous to FAK, at the 402 phosphorylation site and promoted its association to FAK in a time-dependent manner. Mutation of PYK2 at the Tyr-402 site prevented the ERK2 phosphorylation only at 4 h. Intra and extracellular calcium chelators prevented PYK2 activation only at 4 h. In summary, our data showed that osteoblast response to mitogenic CS was mediated by MEK pathway activation. The latter was induced by ERK2 phosphorylation under the control of FAK and PYK2 phosphorylation orchestrated in a time-dependent manner. It is now well evidenced that mechanical stimulation can increase bone mass in vivo (1Oxlund H. Andersen N.B. Ortoft G. Orskov H. Andreassen T.T. Endocrinology. 1998; 139: 1899-1904Crossref PubMed Scopus (43) Google Scholar) through an increase in the number of osteoblasts, the bone-forming cells. In vitro experiments have revealed that physical forces act directly at the cellular level and showed that bone cells are able to respond physiologically to mechanical stress (2Banes A.J. Tsuzaki M. Yamamoto J. Fischer T. Brigman B. Brown T. Miller L. Cell Biol. 1995; 73: 349-365Google Scholar, 3Wozniak M. Fausto A. Carron C.P. Meyer D.M. Hruska K.A. J. Bone Miner. Res. 2000; 15: 1731-1745Crossref PubMed Scopus (119) Google Scholar). It was shown that cyclic mechanical loading is more effective in inducing osteoblast proliferation and gene expression than is static loading (4Damien E. Price J.S. Lanyon L.E. J. Bone Miner. Res. 2000; 15: 2169-2177Crossref PubMed Scopus (146) Google Scholar). There is a large body of evidence showing that extracellular signal-regulated kinase 1/2 (ERK1/2) 1The abbreviations used are: ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FCS, fetal calf serum; HA, hemagglutinin A; MAPK mitogen-activated protein kinase; PBS, phosphate-buffered saline; PI, proliferation index; PYK2, proline-rich tyrosine kinase 2; BAPTA-AM, 1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester. controls cell proliferation in various cell types, including osteoblasts (5Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Recent reports demonstrated that mechanical strain is able to activate ERK1/2 in several adherent cells such as mesangial cells (6Ingram A.J. James L. Cai L. Thai K. Ly H. Scholey J.W. J. Biol. Chem. 2000; 275: 40301-40306Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), smooth muscle cells (7Numaguchi K. Eguchi S. Yamakawa T. Motley E.D. Inagami T. Circ. Res. 1999; 85: 5-11Crossref PubMed Scopus (186) Google Scholar), and chondrocytes (8Fanning P.J. Emkey G. Smith R.J. Grodzinsky A.J. Szasz N. Trippel S.B. J. Biol. Chem. 2003; 278: 50940-50948Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In osteoblasts, ERK activation was mostly reported in fluid flow (9You J. Reilly G.C. Zhen X. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. 2000; PubMed Scopus Google Scholar, S. 2003; PubMed Scopus Google Scholar, J. H. S. J. PubMed Scopus Google Scholar), M. C.F. J. Bone Miner. Res. 2003; PubMed Scopus Google Scholar), H. B. J. J. Bone Miner. Res. PubMed Scopus Google Scholar) experiments and in only one cyclic stretch with fluid Lanyon L.E. PubMed Scopus Google Scholar). cell stretch to a of mechanical in the bone N. PubMed Scopus Google Scholar, A. K. M. T. K. K. M. K. T. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) and J. Google Scholar). The mechanisms that control induced ERK activation in osteoblasts remain are as in mechanical ERK activation H. B. J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. J. 2003; Google Scholar). cell such as tyrosine phosphorylation calcium and mitogen-activated protein kinase J.S. 1995; PubMed Scopus Google Scholar, P.J. PubMed Scopus Google Scholar). the was to ERK1/2 activation mediated in a cyclic strain cell In we on of activated by mechanical adhesion kinase (FAK) is a protein tyrosine kinase that reported to an in P.J. PubMed Scopus Google Scholar). showed that the adhesion of cells of the extracellular the activation of such as FAK at including Tyr-397 P.J. Biol. 1999; PubMed Scopus Google Scholar, Biol. 1995; 15: PubMed Google Scholar), the only site Biol. PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, E. Cell Biol. 2000; PubMed Scopus Google Scholar). This is a site the Src 2 of Src family leading to a FAK and Src family Biol. 1995; 15: PubMed Google Scholar, Biol. 1999; PubMed Scopus Google Scholar, J.W. Biol. PubMed Scopus Google Scholar). This association of with the one of activation of ERK by FAK T. PubMed Scopus Google Scholar). This of phosphorylation events and was reported is not events are induced by the mechanical strain to adherent cells. tyrosine we expression of the adhesion focal tyrosine also as proline-rich tyrosine kinase 2 S. H. E. B. J. 1995; PubMed Scopus Google Scholar), cell adhesion kinase H. K. M. H. K. T. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), kinase H. X. B. M. R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) is to as PYK2 a with FAK and of the FAK tyrosine phosphorylation and are at in PYK2 and PYK2 was to activated in osteoblasts by a bone M. M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that this tyrosine kinase a in the stimulation of osteoblast proliferation M. L. J. Bone Miner. Res. PubMed Scopus Google Scholar). It shown that the tyrosine phosphorylation of PYK2 was the is S. H. E. B. J. 1995; PubMed Scopus Google Scholar). In our study, we to the of in PYK2 activation by mechanical PYK2 and FAK were by of and were and the and were were The was ERK2 and FAK were 2 and inhibitor were and were used ROS 17/2.8 osteoblast-like a well cell R.J. S.B. Endocrinology. PubMed Scopus Google Scholar) with the of osteoblasts and type I and the to and the cell of cells after to were to with in at with was with with fetal calf Cell was then at and in with and 2 The was after the h to cells. were in of at ROS 17/2.8 cells were in and cells were in with FCS, 2 and a cells were with and plated type I was used the of mechanical stretch to osteoblasts A.J. J. J. Cell PubMed Google Scholar). were plated in with type I silicone at cells well proliferation and cells well after cells were serum-starved overnight and then to mechanical was induced with a strain of a by that are by a The the loading that of that a of the strain of the of the experiments a of was through an to the were to a of and then the were to The used in this 1% at a of period by a and the of the is directly to cells. cells on were used as 17/2.8 cell proliferation was with N. A. E. B. N. Biol. 1995; Google Scholar, Cell Res. 2003; PubMed Scopus Google Scholar) to the is a that the cell and is to cells It was shown that is and and on cell proliferation J. K.A. M. M. X. Res. 1998; Google Scholar). Cell was in and in 4 at The was by the of 2 of and 4 of proliferation were at and with to cells the cells were at cells type I membranes. was at 0 in and at 4 analysis as a 0 cells were to h after were then in overnight and then to static strained as in the in the of 30 the mechanical A. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were in and at 4 was on a cell with a the Cell analysis was on a of cells. of the cell was the Cell on the of cells in the proliferation and the of by a The PI, a of the proliferation of cell was by in is the of the in and the the of the as by is the number of cells in the with is the and is the are of a of experiments in with and were in 1% 1% and were by at 30 were to of was by the acid were by of cell and 2 of FAK, PYK2, and at 4 2 h on a were at 4 overnight on a by protein were with in and by min, by and then by Western Western were to were blocked in PBS, and overnight at 4 with the and of were used at a of were with and then with h at with with the were were by and with were by were to mechanical and protein tyrosine phosphorylation was by immunofluorescence cells were h on type I were then in overnight and then to static mechanical strain as in the was on and control cells at various after the of the were in min, with PBS, and with in 2 in 1% and in 1% were h at and the cells were in on a were with in at of tyrosine residues were with and with a and were with a on cells was on of phosphotyrosine are reported in A. N. L. PubMed Scopus Google Scholar). were by the analysis the number of A. N. L. PubMed Scopus Google Scholar). and control mutated PYK2 and the mutated FAK were in cells on and then in was at were by the and and the of and was assessed by 1% of was was at in control and the were ROS 17/2.8 cells with to the cells were and to were then in overnight and then to static strained as in the were then in were by by with of the cells were highly as by also we ROS 17/2.8 with and then with the data were at experiments in were as were with of was this ROS 17/2.8 cell proliferation was and h after strain shown in the of cells in can assessed by the PI, a of the proliferation of cell ROS 17/2.8 cells to cyclic strain were more than cells at h as well as at h after a strain period in a as shown by both the and the of cells in the strained cell were and in the and the the cell were and in the and the MEK that the mitogenic cyclic strain was mediated by MEK pathway activation. Treatment of the with the MEK inhibitor the period did not the cell at not at the completely blocked static in the to static in control increase in proliferation was in response to strain on proliferation was with the cyclic strain stimulated ROS 17/2.8 we ERK phosphorylation in experiments strain induced ERK2 phosphorylation in a time-dependent in to the with was ERK2 at Tyr-187 phosphorylation 0 to 4 h after strain to of control as by of by the of protein tyrosine phosphorylation involved in ERK tyrosine phosphorylation of cellular was by an In response to cyclic a time-dependent tyrosine phosphorylation of several was in ROS 17/2.8 cells as well as in the cell not of and The increase in the tyrosine phosphorylation level was as as the of strain and was to 4 h. after an overnight a level of protein tyrosine phosphorylation was of protein tyrosine phosphorylation to the extracellular adhesion K. J. Cell Biol. PubMed Scopus Google Scholar). were at and we the of the tyrosine of S. A. PubMed Scopus Google Scholar), Src J. Cell PubMed Google Scholar), and PYK2 X. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of by was of static and cells with the and with the the The level of tyrosine phosphorylation at FAK-Tyr-397 and cells was as with that of static cells. were in cells as well as in cells and cells FAK-Tyr-397 phosphorylation in a time-dependent 30 after strain and at the level at 4 h after strain of static a ROS 17/2.8 cell transfection with FAK mutated at Tyr-397 the phosphorylation of ERK2 at Tyr-187 transfection with an did not prevent the ERK2 phosphorylation at the in cells phosphorylation at Tyr-397 in response to cyclic strain on cells and cells plated on were to strain 0 were 30 min, 4 h after the of were with an FAK were by by of and with an The was then and with an transfection with FAK mutated at the phosphorylation of ERK2 at ROS 17/2.8 cells were with the control were with an were by by the of and with an the were with a MAPK and protein loading was by the and with an of Src by and the of Src in FAK in to family are tyrosine phosphorylation of FAK Biol. 1999; PubMed Scopus Google Scholar). of Src was static and cell by by the of and with an induced a increase in the level of tyrosine phosphorylation of Src at as with static cells This increase as as the of stretch and to 4 h after of Src with FAK showed that the was by strain as as the of strain and to 4 h the of Src family in the phosphorylation of FAK at we the of pyrazolopyrimidine a selective inhibitor of Src family kinase J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), on FAK Tyr-397 phosphorylation induced by mechanical cells were with (10 30 a that was shown to Src family activation E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Cell were with an and the were by by Western blotting with a the of FAK Treatment with the phosphorylation at Tyr-397 in did not prevent the FAK phosphorylation at Tyr-397 induced by mechanical suggesting a Src-independent activation of FAK. In the increase in activation at 4 h. of Src family in FAK phosphorylation at Tyr-397 in response to cyclic plated on were 30 in the of (10 a stretch were 30 min, 4 h after the of were with an FAK were by by of and with an The was then and with an an in and is well in cell such as smooth muscle cells that are mechanical is to activate FAK in smooth muscle cells T. Circ. Res. PubMed Scopus Google Scholar, N. K. T. Res. 1999; PubMed Scopus Google Scholar), we that the tyrosine phosphorylation events we at focal the of cells were to a and by phosphotyrosine residues a and tyrosine phosphorylation of FAK at Tyr-397 a FAK-Tyr-397 not cells were In and FAK-Tyr-397 cell to focal adhesion was more in strained cells as with control cells. This was by the phosphotyrosine number was of static control after and the phosphotyrosine a increase at 2 h of PYK2 by and shown to an phosphorylation in and cells J. H. S. S. PubMed Google Scholar, A. H. J. T. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and was shown to activated by mechanical strain in smooth muscle cells H. T. T. Endocrinology. 2003; PubMed Scopus Google Scholar). The level of PYK2 tyrosine phosphorylation at Tyr-402 cells at 30 with a at 4 h increase as with static Cell transfection with PYK2 mutated at Tyr-402 ERK2 tyrosine phosphorylation in PYK2 phosphorylation of ERK2 at Tyr-187 induced by a PYK2 mechanical strain activation 4 h after strain of PYK2 with FAK showed that the association with strain in a time-dependent PYK2 with in to the with FAK, this association was not by strain transfection with PYK2 mutated at Tyr-402 the phosphorylation of ERK2 at ROS 17/2.8 cells were with control were with an were by by of and with an were with the MAPK protein loading was by the and with an of FAK with PYK2 in a time-dependent manner. ROS 17/2.8 cells were PYK2 The were then with It shown that the of calcium is PYK2 activation S. H. E. B. J. 1995; PubMed Scopus Google Scholar) and is involved in mechanical Reilly 1995; Google Scholar). of to the activation of PYK2, we the phosphorylation of PYK2 after with the strain activation of PYK2 at a increase was under mechanical strain as with the with an that the PYK2 phosphorylation and prevented the at 4 h after did not the at 30 extracellular by phosphorylation of PYK2 at Tyr-402 at 30 min, was 4 h after data that PYK2 activation was on both and extracellular a in the of a of cell types, including smooth muscle cells H. T. T. Endocrinology. 2003; PubMed Scopus Google Scholar), cells M. T. J. Res. PubMed Scopus Google Scholar, M. S. S. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Circ. Res. PubMed Google Scholar), and bone cells Res. PubMed Scopus Google Scholar). It the of a a response leading to both in gene expression as well as in In the of a mechanical strain at in vivo was shown to increase bone mass through an increase in formation to a stimulation of cell S. S. J. Bone Miner. Res. 1995; PubMed Scopus Google Scholar). showed that have to the of proliferation in response to various in several cell types, including osteoblasts (5Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, Res. PubMed Scopus Google Scholar, S. M. B. J. 2000; PubMed Scopus Google Scholar). strain is to activate ERK1/2 and the and of activation of MAPK to with cell and the A.J. Ly H. Thai K. Scholey J.W. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. T. J. 1999; 276: Scholar, K. H. J. 1998; PubMed Scopus Google Scholar). In the study, we that a stretch period was able to increase ROS 17/2.8 cell proliferation after h in were in cells after a period of in the of A. A. N. L. Cell 2003; PubMed Google Scholar). experiments were with cyclic strain at 1% was the of the as in the of A. A. M. M. E. J. Bone 2000; Scopus Google Scholar). The of cyclic strain on ROS 17/2.8 proliferation were blocked by a MEK inhibitor that and ERK2 activation. have the used is with that used to ERK activation A. A. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1999; PubMed Scopus Google Scholar). this did not the proliferation as with The ERK pathway reported as activated by mechanical in of the used fluid flow (9You J. Reilly G.C. Zhen X. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. J. 2000; PubMed Scopus Google Scholar, S. 2003; PubMed Scopus Google Scholar, J. H. S. J. PubMed Scopus Google Scholar) to stress on bone cells. It is not well the bone cell response to mechanical is J. Google Scholar) showed that bone cells and under the cells did not respond to stretch in that ERK activation both Lanyon L.E. PubMed Scopus Google Scholar). In also used as a mechanical in did ERK activation in FAK phosphorylation did not in this M. C.F. J. Bone Miner. Res. 2003; PubMed Scopus Google Scholar), was in our data various to in of bone are not in of events the type and of also induced ERK activation. N. N. K. M. Res. 1998; PubMed Scopus Google Scholar) reported in ERK phosphorylation after stretch in in this latter the strain was than that used in our with extracellular a in that control cell and the one the family of cell cell with the extracellular is the formation of focal adhesion including the of FAK. This is by the activation of the ERK pathway. the focal have as a site the cellular of mechanical forces in a of cell B. A. Cell Biol. 2001; PubMed Scopus Google Scholar). mechanical stretch the ERK pathway through with FAK in cells J. A. J. 2003; Google Scholar). the and ERK activation induced by mechanical strain in osteoblasts are not In this we showed the that mechanical strain rapidly activated FAK-Tyr-397 phosphorylation by phosphorylation in we a FAK-Tyr-397 phosphorylation in osteoblasts and that cells are a data are in with reports that showed a mechanical FAK-Tyr-397 phosphorylation in vitro and in vivo M. S. A. 2001; PubMed Scopus Google Scholar, Circ. Res. 2003; PubMed Scopus Google Scholar) in and in the FAK at tyrosine is by phosphorylation by tyrosine at including to the activation of the pathway T. PubMed Scopus Google Scholar). It shown that ERK activation can of FAK through phosphorylation L. G. E. M. G. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). in our study, transfection with FAK mutated at Tyr-397 phosphorylation of ERK2 at that FAK-Tyr-397 phosphorylation is strain activation of The phosphorylation of FAK at Tyr-397 the formation of with including Src family Biol. PubMed Scopus Google Scholar), the H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the X. A. P.J. G. S. A. 1999; PubMed Scopus Google Scholar), the J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and T. Biol. 1998; PubMed Scopus Google Scholar), and the M. J. T. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of FAK at Tyr-397 is the of a site the Src 2 of Src family J.W. Biol. PubMed Scopus Google Scholar). In this study, mechanical stimulation induced an increase in the control as as the of strain that to 4 h. we that with a selective inhibitor of Src family the phosphorylation at Tyr-397 in as reported E. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). did not prevent mechanical FAK-Tyr-397 activation at 30 the activation at 4 h. our data that the of phosphorylation 4 h after mechanical strain are with the that association can to the of to the with the of FAK-Tyr-397 phosphorylation by at such as Biol. 1999; PubMed Scopus Google Scholar). It is well evidenced that FAK phosphorylation to extracellular cell adhesion P.J. Biol. 1999; PubMed Scopus Google Scholar, Biol. 1995; 15: PubMed Google Scholar). the of the FAK phosphorylation in adherent cells that are to mechanical strain showed that with and to cells to FAK activation H. B. J. J. Bone Miner. Res. PubMed Scopus Google Scholar). In the study, of phosphotyrosine residues to focal adhesion was more in strained with control cells as well as This was by of phosphotyrosine number and number increase as as the of suggesting that strain rapidly induced formation of focal In the was only at 4 suggesting that by the of the focal was a in osteoblast cell adhesion to a mechanical K. H. M. J. Cell 2001; Google Scholar). In to the on the Western of strained cell we a protein that we as PYK2, a protein highly homologous to FAK, only reported as involved in cell response to mechanical stretch in smooth muscle cells H. T. T. Endocrinology. 2003; PubMed Scopus Google Scholar). M. H. J. M. PubMed Scopus Google Scholar) reported that is able to cell induced both FAK and PYK2 suggesting that tyrosine a in the of in the of mechanical strain on bone to reported that induced a PYK2 Src association M. M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), in our association after and such increase was Src and PYK2 In our study, the of PYK2 and FAK tyrosine phosphorylation with a PYK2 at 4 suggesting that the activation of tyrosine not mediated by the PYK2 to strain activation of in to the with FAK, cell transfection with PYK2 mutated at Tyr-402 the phosphorylation of ERK2 at The ERK2 mechanical phosphorylation at 30 In the at 4 h was completely the of PYK2 in ERK activation. FAK and PYK2 both ERK phosphorylation stimulated by mechanical shown that PYK2 directly FAK, the that Src are on FAK X. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In FAK PYK2 activation by a kinase we that mechanical strain the association of FAK with PYK2 in a time-dependent manner. It was shown cell of to the of FAK and PYK2 to the focal adhesion and the phosphorylation of a of S. M. S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). PYK2 activation shown that this tyrosine kinase an phosphorylation in and J. H. S. S. PubMed Google Scholar, A. H. J. T. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A.J. L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). a in calcium was reported to the of PYK2 Reilly 1995; Google Scholar). In calcium are to the in stimulated bone cells S. H. E. B. J. 1995; PubMed Scopus Google Scholar). In the study, we showed that both with PYK2 activation 4 h after suggesting that PYK2 phosphorylation at this both calcium and BAPTA-AM, a cell calcium PYK2 activation at Tyr-402 in a than that with in smooth muscle cells the PYK2 activation induced by mechanical stretch was prevented by a inhibitor H. T. T. Endocrinology. 2003; PubMed Scopus Google Scholar). In (9You J. Reilly G.C. Zhen X. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) that the increase in cells under fluid flow was completely after the of an calcium In the of in response to stretch in osteoblasts A.J. J. 2000; PubMed Scopus Google Scholar). our calcium a in PYK2 is that involved in PYK2 activation not on In we that mitogenic mechanical strain the of tyrosine phosphorylation at focal through an increase in both the number and size of adhesion in cells. strain promoted the association of the highly homologous tyrosine FAK and PYK2 and with tyrosine were both the activation of Src family FAK calcium were PYK2 activation. in data that both PYK2 and FAK the events cell induced by mechanical the mechanisms involved in FAK and PYK2 activation to of of mechanical strain on the