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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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Tubulin-bound and  nonbound c-src in adherent  cells. The lysate of adherent  avian marrow macrophages  was immunoprecipitated with  antitubulin antibody (lane 1),  and the supernatant was reimmunoprecipitated with the  same antibody (lane 2). The  supernatant, now depleted of  tubulin-associated c-src, was immunoprecipitated with anti–c-src  antibody (lane 3). All lanes were probed with anti–c-src antibody.
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Figure 4: Tubulin-bound and nonbound c-src in adherent cells. The lysate of adherent avian marrow macrophages was immunoprecipitated with antitubulin antibody (lane 1), and the supernatant was reimmunoprecipitated with the same antibody (lane 2). The supernatant, now depleted of tubulin-associated c-src, was immunoprecipitated with anti–c-src antibody (lane 3). All lanes were probed with anti–c-src antibody.

Mentions: Although unlikely, considering the short duration of substrate attachment, the differences in c-src/tubulin association between adherent and nonadherent macrophages might reflect adhesion-induced expression of either protein. Thus, we compared the quantities of both proteins in suspended and plated cells by immunoprecipitating cell lysates with excess anti–c-src or antitubulin and immunoblotting the respective product with the same antibody. As seen in Fig. 3, B and C, nonadherent and adherent macrophages contain the same amounts of both proteins. For reasons unknown, we consistently find c-src migrates slower when immunoprecipitated with antitubulin antibody as compared to its direct immunoprecipitation (IP) with anti–c-src antibody (e.g., see Figs. 4 and 14). In the conditions of these experiments, c-src immunoblots are within the linear range of analysis (see Fig. 2).


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)

Tubulin-bound and  nonbound c-src in adherent  cells. The lysate of adherent  avian marrow macrophages  was immunoprecipitated with  antitubulin antibody (lane 1),  and the supernatant was reimmunoprecipitated with the  same antibody (lane 2). The  supernatant, now depleted of  tubulin-associated c-src, was immunoprecipitated with anti–c-src  antibody (lane 3). All lanes were probed with anti–c-src antibody.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Tubulin-bound and nonbound c-src in adherent cells. The lysate of adherent avian marrow macrophages was immunoprecipitated with antitubulin antibody (lane 1), and the supernatant was reimmunoprecipitated with the same antibody (lane 2). The supernatant, now depleted of tubulin-associated c-src, was immunoprecipitated with anti–c-src antibody (lane 3). All lanes were probed with anti–c-src antibody.
Mentions: Although unlikely, considering the short duration of substrate attachment, the differences in c-src/tubulin association between adherent and nonadherent macrophages might reflect adhesion-induced expression of either protein. Thus, we compared the quantities of both proteins in suspended and plated cells by immunoprecipitating cell lysates with excess anti–c-src or antitubulin and immunoblotting the respective product with the same antibody. As seen in Fig. 3, B and C, nonadherent and adherent macrophages contain the same amounts of both proteins. For reasons unknown, we consistently find c-src migrates slower when immunoprecipitated with antitubulin antibody as compared to its direct immunoprecipitation (IP) with anti–c-src antibody (e.g., see Figs. 4 and 14). In the conditions of these experiments, c-src immunoblots are within the linear range of analysis (see Fig. 2).

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