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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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C-src associates with tubulin in a substrate adhesion– dependent manner. Avian marrow macrophages were maintained, nonadherent, in Teflon beakers for 2 d. The cells were  then plated on tissue culture plastic (adherent) or retained in Teflon beakers (nonadherent) for an additional 90 min. Adherent or  nonadherent cells were lysed and immunoprecipitated with anti– c-src or antitubulin antibodies. The immunoprecipitates were  separated by nonreducing 8% SDS-PAGE. The proteins were  transferred to a nitrocellulose membrane and probed with anti–csrc or antitubulin antibodies, followed by ECL detection, using  HRP-conjugated goat anti–mouse IgG antibody. A represents  the entire gel.
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Figure 3: C-src associates with tubulin in a substrate adhesion– dependent manner. Avian marrow macrophages were maintained, nonadherent, in Teflon beakers for 2 d. The cells were then plated on tissue culture plastic (adherent) or retained in Teflon beakers (nonadherent) for an additional 90 min. Adherent or nonadherent cells were lysed and immunoprecipitated with anti– c-src or antitubulin antibodies. The immunoprecipitates were separated by nonreducing 8% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and probed with anti–csrc or antitubulin antibodies, followed by ECL detection, using HRP-conjugated goat anti–mouse IgG antibody. A represents the entire gel.

Mentions: To further characterize the putative tubulin and c-src proteins present in marrow macrophage lysate, and to determine if they associate in a cell-matrix–dependent manner, we performed c-src immunoblots on immunoprecipitated tubulin. To this end, chicken marrow macrophages were maintained, nonadherent, for 2 d, after which they were transferred to serum-free medium. One half the cells were kept nonadherent, and the other half were plated on tissue culture plastic, to which they rapidly attach. 90 min later both samples were lysed with digitonin in conditions known to preserve microtubule architecture (51). The lysate was immunoprecipitated with an antitubulin antibody and the immunoprecipitate c-src content assessed by immunoblot. Fig. 3 A shows minimal c-src precipitates with tubulin in nonadherent cells, which typically exhibit little cytoskeletal organization. In contrast, abundant c-src associates with tubulin precipitated from macrophages adherent for 90 min, a period sufficient to permit microtubule formation (infra vide). Importantly, while a number of slower migrating bands are present in both immunoprecipitates, only the quantity of c-src is regulated by substrate adherence.


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)

C-src associates with tubulin in a substrate adhesion– dependent manner. Avian marrow macrophages were maintained, nonadherent, in Teflon beakers for 2 d. The cells were  then plated on tissue culture plastic (adherent) or retained in Teflon beakers (nonadherent) for an additional 90 min. Adherent or  nonadherent cells were lysed and immunoprecipitated with anti– c-src or antitubulin antibodies. The immunoprecipitates were  separated by nonreducing 8% SDS-PAGE. The proteins were  transferred to a nitrocellulose membrane and probed with anti–csrc or antitubulin antibodies, followed by ECL detection, using  HRP-conjugated goat anti–mouse IgG antibody. A represents  the entire gel.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: C-src associates with tubulin in a substrate adhesion– dependent manner. Avian marrow macrophages were maintained, nonadherent, in Teflon beakers for 2 d. The cells were then plated on tissue culture plastic (adherent) or retained in Teflon beakers (nonadherent) for an additional 90 min. Adherent or nonadherent cells were lysed and immunoprecipitated with anti– c-src or antitubulin antibodies. The immunoprecipitates were separated by nonreducing 8% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane and probed with anti–csrc or antitubulin antibodies, followed by ECL detection, using HRP-conjugated goat anti–mouse IgG antibody. A represents the entire gel.
Mentions: To further characterize the putative tubulin and c-src proteins present in marrow macrophage lysate, and to determine if they associate in a cell-matrix–dependent manner, we performed c-src immunoblots on immunoprecipitated tubulin. To this end, chicken marrow macrophages were maintained, nonadherent, for 2 d, after which they were transferred to serum-free medium. One half the cells were kept nonadherent, and the other half were plated on tissue culture plastic, to which they rapidly attach. 90 min later both samples were lysed with digitonin in conditions known to preserve microtubule architecture (51). The lysate was immunoprecipitated with an antitubulin antibody and the immunoprecipitate c-src content assessed by immunoblot. Fig. 3 A shows minimal c-src precipitates with tubulin in nonadherent cells, which typically exhibit little cytoskeletal organization. In contrast, abundant c-src associates with tubulin precipitated from macrophages adherent for 90 min, a period sufficient to permit microtubule formation (infra vide). Importantly, while a number of slower migrating bands are present in both immunoprecipitates, only the quantity of c-src is regulated by substrate adherence.

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