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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 specific extracellular matrix proteins on c-src/ tubulin association. Avian marrow macrophages, incubated in  Teflon beakers for 2 d, were plated on untreated tissue culture  plastic or that coated with indicated extracellular matrix proteins.  After 90 min, the cells were lysed, and the lysate was immunoprecipitated with antitubulin antibody. C-src content of immunoprecipitates was assessed by immunoblot as described in Fig. 3.
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Figure 12: Effect of specific extracellular matrix proteins on c-src/ tubulin association. Avian marrow macrophages, incubated in Teflon beakers for 2 d, were plated on untreated tissue culture plastic or that coated with indicated extracellular matrix proteins. After 90 min, the cells were lysed, and the lysate was immunoprecipitated with antitubulin antibody. C-src content of immunoprecipitates was assessed by immunoblot as described in Fig. 3.

Mentions: The substrate binding experiments described thus far were performed in serum-free medium devoid of potential extracellular attachment molecules, a nonphysiological circumstance. Thus, we asked if c-src/tubulin association is prompted by specific extracellular matrix proteins. To this end, cells were maintained, serum free, for 90 min in suspension or plastic dishes, the latter either untreated or coated with fibronectin, vitronectin, or type I collagen. The macrophages were harvested in lysis buffer and lysates immunoprecipitated with antitubulin antibody. Fig. 12 shows that when analyzed by immunoblot, antitubulin immunoprecipitates, derived from cells adherent to fibronectin or vitronectin, contain as much c-src as do those plated on untreated plastic. In contrast, plating on type I collagen yields no detectable c-src/tubulin association.


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 specific extracellular matrix proteins on c-src/ tubulin association. Avian marrow macrophages, incubated in  Teflon beakers for 2 d, were plated on untreated tissue culture  plastic or that coated with indicated extracellular matrix proteins.  After 90 min, the cells were lysed, and the lysate was immunoprecipitated with antitubulin antibody. C-src content of immunoprecipitates was assessed by immunoblot as described in Fig. 3.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 12: Effect of specific extracellular matrix proteins on c-src/ tubulin association. Avian marrow macrophages, incubated in Teflon beakers for 2 d, were plated on untreated tissue culture plastic or that coated with indicated extracellular matrix proteins. After 90 min, the cells were lysed, and the lysate was immunoprecipitated with antitubulin antibody. C-src content of immunoprecipitates was assessed by immunoblot as described in Fig. 3.
Mentions: The substrate binding experiments described thus far were performed in serum-free medium devoid of potential extracellular attachment molecules, a nonphysiological circumstance. Thus, we asked if c-src/tubulin association is prompted by specific extracellular matrix proteins. To this end, cells were maintained, serum free, for 90 min in suspension or plastic dishes, the latter either untreated or coated with fibronectin, vitronectin, or type I collagen. The macrophages were harvested in lysis buffer and lysates immunoprecipitated with antitubulin antibody. Fig. 12 shows that when analyzed by immunoblot, antitubulin immunoprecipitates, derived from cells adherent to fibronectin or vitronectin, contain as much c-src as do those plated on untreated plastic. In contrast, plating on type I collagen yields no detectable c-src/tubulin association.

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