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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 temperature on tubulin/c-src association. Avian marrow macrophages, incubated in suspension for 2 d, were retained  nonadherent or plated on tissue culture plastic for 90 min. (A) Cells were lysed with either ice-cold or warm (37°C) lysis buffer and the  lysate immunoprecipitated with antitubulin or anti–c-src antibodies at the same respective temperatures. In part B, ice-cold prepared lysates were divided equally; one half was rewarmed and maintained at 37°C in the presence of GTP for 30 min, and the other half was  kept at 4°C. Lysates were immunoprecipitated and c-src content of the immunoprecipitates was assessed by immunoblot as described in  Fig. 3.
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Figure 9: Effect of temperature on tubulin/c-src association. Avian marrow macrophages, incubated in suspension for 2 d, were retained nonadherent or plated on tissue culture plastic for 90 min. (A) Cells were lysed with either ice-cold or warm (37°C) lysis buffer and the lysate immunoprecipitated with antitubulin or anti–c-src antibodies at the same respective temperatures. In part B, ice-cold prepared lysates were divided equally; one half was rewarmed and maintained at 37°C in the presence of GTP for 30 min, and the other half was kept at 4°C. Lysates were immunoprecipitated and c-src content of the immunoprecipitates was assessed by immunoblot as described in Fig. 3.

Mentions: To further confirm the role of microtubule formation in c-src/tubulin association, we exposed macrophages to cold, also known to inhibit tubulin polymerization within solution or cells (9, 29, 54). Thus, adherent or nonadherent cells were lysed and immunoprecipitated either at 4 or 37°C. While the protein complex is maintained in adherent cells at 37°C, little detectable c-src associates (<5% compared to c-src in adherent cells at 37°C) with tubulin at 4°C (Fig. 9 A). To determine if this phenomenon may be biologically significant, we asked if loss of c-src/tubulin association, induced by cold, is reversible by rewarming, a circumstance that repolymerizes tubulin (14). As seen in Fig. 9 B, coimmunoprecipitation of the two proteins is reestablished upon warming 4°C-exposed cells.


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 temperature on tubulin/c-src association. Avian marrow macrophages, incubated in suspension for 2 d, were retained  nonadherent or plated on tissue culture plastic for 90 min. (A) Cells were lysed with either ice-cold or warm (37°C) lysis buffer and the  lysate immunoprecipitated with antitubulin or anti–c-src antibodies at the same respective temperatures. In part B, ice-cold prepared lysates were divided equally; one half was rewarmed and maintained at 37°C in the presence of GTP for 30 min, and the other half was  kept at 4°C. Lysates were immunoprecipitated and c-src content of the immunoprecipitates was assessed by immunoblot as described in  Fig. 3.
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Related In: Results  -  Collection

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

Figure 9: Effect of temperature on tubulin/c-src association. Avian marrow macrophages, incubated in suspension for 2 d, were retained nonadherent or plated on tissue culture plastic for 90 min. (A) Cells were lysed with either ice-cold or warm (37°C) lysis buffer and the lysate immunoprecipitated with antitubulin or anti–c-src antibodies at the same respective temperatures. In part B, ice-cold prepared lysates were divided equally; one half was rewarmed and maintained at 37°C in the presence of GTP for 30 min, and the other half was kept at 4°C. Lysates were immunoprecipitated and c-src content of the immunoprecipitates was assessed by immunoblot as described in Fig. 3.
Mentions: To further confirm the role of microtubule formation in c-src/tubulin association, we exposed macrophages to cold, also known to inhibit tubulin polymerization within solution or cells (9, 29, 54). Thus, adherent or nonadherent cells were lysed and immunoprecipitated either at 4 or 37°C. While the protein complex is maintained in adherent cells at 37°C, little detectable c-src associates (<5% compared to c-src in adherent cells at 37°C) with tubulin at 4°C (Fig. 9 A). To determine if this phenomenon may be biologically significant, we asked if loss of c-src/tubulin association, induced by cold, is reversible by rewarming, a circumstance that repolymerizes tubulin (14). As seen in Fig. 9 B, coimmunoprecipitation of the two proteins is reestablished upon warming 4°C-exposed cells.

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