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Substrate recognition by osteoclast precursors induces C-src/microtubule association.

Abu-Amer Y, Ross FP, Schlesinger P, Tondravi MM, Teitelbaum SL - J. Cell Biol. (1997)

Bottom Line: In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate.The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity.Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.

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Effect of microtubule association/dissociation on c-src  kinase activity. Adherent macrophages were lysed at 37°C (to maintain intact microtubules) or at 4°C. Lysates were immunoprecipitated with antitubulin antibody (lanes 1 and 3) followed by  immunoprecipitation with anti–c-src antibody (lanes 2 and 4, respectively). Immunoprecipitates were divided equally. One half  was subjected to in vitro kinase assay (A) and the other half to  Western immunoblot analysis with anti–c-src antibody. B represents the specific activity determined from densitometric analysis  of the ratio between c-src kinase activity and c-src protein in each  condition.
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Figure 11: Effect of microtubule association/dissociation on c-src kinase activity. Adherent macrophages were lysed at 37°C (to maintain intact microtubules) or at 4°C. Lysates were immunoprecipitated with antitubulin antibody (lanes 1 and 3) followed by immunoprecipitation with anti–c-src antibody (lanes 2 and 4, respectively). Immunoprecipitates were divided equally. One half was subjected to in vitro kinase assay (A) and the other half to Western immunoblot analysis with anti–c-src antibody. B represents the specific activity determined from densitometric analysis of the ratio between c-src kinase activity and c-src protein in each condition.

Mentions: We have shown c-src kinase activity preferentially associates with tubulin derived from adherent rather than nonadherent cells (Fig. 7). To determine if the low level of tubulinbound c-src activity in nonadherent macrophages reflects enzymatic repression by globular tubulin, we assessed specific activity of the tyrosine kinase in lysates derived from adherent cells maintained at 37 or 4°C, respectively. Thus, lysates of cells cultured at either temperature were immunoprecipitated with antitubulin and then anti–c-src antibodies. Equal amounts of protein were subjected to in vitro kinase assay or subjected to immunoblot with anti–c-src antibody. Once again, c-src activity is tubulin associated at 37 but not at 4°C (Fig. 11). Establishing the absence of microtubules does not repress the tyrosine kinase, non–tubulin-bound c-src–specific activity is similar in lysates derived from macrophages cultured at either temperature (compare lanes 2 and 4).


Substrate recognition by osteoclast precursors induces C-src/microtubule association.

Abu-Amer Y, Ross FP, Schlesinger P, Tondravi MM, Teitelbaum SL - J. Cell Biol. (1997)

Effect of microtubule association/dissociation on c-src  kinase activity. Adherent macrophages were lysed at 37°C (to maintain intact microtubules) or at 4°C. Lysates were immunoprecipitated with antitubulin antibody (lanes 1 and 3) followed by  immunoprecipitation with anti–c-src antibody (lanes 2 and 4, respectively). Immunoprecipitates were divided equally. One half  was subjected to in vitro kinase assay (A) and the other half to  Western immunoblot analysis with anti–c-src antibody. B represents the specific activity determined from densitometric analysis  of the ratio between c-src kinase activity and c-src protein in each  condition.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139850&req=5

Figure 11: Effect of microtubule association/dissociation on c-src kinase activity. Adherent macrophages were lysed at 37°C (to maintain intact microtubules) or at 4°C. Lysates were immunoprecipitated with antitubulin antibody (lanes 1 and 3) followed by immunoprecipitation with anti–c-src antibody (lanes 2 and 4, respectively). Immunoprecipitates were divided equally. One half was subjected to in vitro kinase assay (A) and the other half to Western immunoblot analysis with anti–c-src antibody. B represents the specific activity determined from densitometric analysis of the ratio between c-src kinase activity and c-src protein in each condition.
Mentions: We have shown c-src kinase activity preferentially associates with tubulin derived from adherent rather than nonadherent cells (Fig. 7). To determine if the low level of tubulinbound c-src activity in nonadherent macrophages reflects enzymatic repression by globular tubulin, we assessed specific activity of the tyrosine kinase in lysates derived from adherent cells maintained at 37 or 4°C, respectively. Thus, lysates of cells cultured at either temperature were immunoprecipitated with antitubulin and then anti–c-src antibodies. Equal amounts of protein were subjected to in vitro kinase assay or subjected to immunoblot with anti–c-src antibody. Once again, c-src activity is tubulin associated at 37 but not at 4°C (Fig. 11). Establishing the absence of microtubules does not repress the tyrosine kinase, non–tubulin-bound c-src–specific activity is similar in lysates derived from macrophages cultured at either temperature (compare lanes 2 and 4).

Bottom Line: In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate.The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity.Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
The osteoclast is distinguished from other macrophage polykaryons by its polarization, a feature induced by substrate recognition. The most striking component of the polarized osteoclast is its ruffled membrane, probably reflecting insertion of intracellular vesicles into the bone apposed plasmalemma. The failure of osteoclasts in c-src-/- osteopetrotic mice to form ruffled membranes indicates pp60(c-src) (c-src) is essential to osteoclast polarization. Interestingly, c-src itself is a vesicular protein that targets the ruffled membrane. This being the case, we hypothesized that matrix recognition by osteoclasts, and their precursors, induces c-src to associate with microtubules that traffic proteins to the cell surface. We find abundant c-src associates with tubulin immunoprecipitated from avian marrow macrophages (osteoclast precursors) maintained in the adherent, but not nonadherent, state. Since the two proteins colocalize only within adherent avian osteoclast-like cells examined by double antibody immunoconfocal microscopy, c-src/tubulin association reflects an authentic intracellular event. C-src/tubulin association is evident within 90 min of cell-substrate recognition, and the event does not reflect increased expression of either protein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous substrate. The increase in microtubule-associated kinase activity attending adhesion mirrors tubulin-bound c-src and does not reflect enhanced specific activity. The fact that microtubule-dissociating drugs, as well as cold, prevent adherence-induced c-src/tubulin association indicates the protooncogene complexes primarily, if not exclusively, with polymerized tubulin. Association of the two proteins does not depend upon protein tyrosine phosphorylation and is substrate specific, as it is induced by vitronectin and fibronectin but not type 1 collagen. Finally, consistent with cotransport of c-src and the osteoclast vacuolar proton pump to the polarized plasmalemma, the H+-ATPase decorates microtubules in a manner similar to the protooncogene, specifically coimmunoprecipitates with c-src from the osteoclast light Golgi membrane fraction, and is present, with c-src, in preparations enriched with acidifying vesicles reconstituted from the osteoclast ruffled membrane.

Show MeSH
Related in: MedlinePlus