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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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Tyrosine kinase activity of tubulin-associated c-src.  One half of avian marrow macrophages maintained nonadherent  for 2 d were plated on tissue culture plastic while the other half  continued in suspension. After 90 min, the cells were lysed, and  the lysate was immunoprecipitated with antitubulin antibody.  One half the immunoprecipitate (A) was assayed for kinase activity by measuring 32P incorporation into c-src and enolase (an exogenous substrate). Tubulin content of the remaining immunoprecipitate was determined by immunoblot (B).
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Figure 7: Tyrosine kinase activity of tubulin-associated c-src. One half of avian marrow macrophages maintained nonadherent for 2 d were plated on tissue culture plastic while the other half continued in suspension. After 90 min, the cells were lysed, and the lysate was immunoprecipitated with antitubulin antibody. One half the immunoprecipitate (A) was assayed for kinase activity by measuring 32P incorporation into c-src and enolase (an exogenous substrate). Tubulin content of the remaining immunoprecipitate was determined by immunoblot (B).

Mentions: To confirm the species immunoprecipitating with tubulin is, in fact, active c-src, we performed in vitro kinase assays on antitubulin immunoprecipitates, using as exogenous substrate, enolase, known to be specifically phosphorylated by the tyrosine kinase. Reflecting the abundance of c-src we find associated with tubulin in adherent osteoclast precursors, Fig. 7 A shows intense autophosphorylation of c-src and phosphorylation of exogenous substrate. In contrast to adherent cells, the magnitude of c-src autophosphorylation is much less pronounced in nonadherent macrophages (18% of the value in adherent cells normalized to protein levels), and phosphorylation of enolase is virtually undetectable. These differences in protein phosphorylation do not reflect altered tubulin abundance in adherent as compared to nonadherent cells (Fig. 7 B). The observed c-src kinase activity parallels, however, the amount of tubulin-associated c-src protein in adherent and nonadherent cells, respectively (Fig. 3 A).


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)

Tyrosine kinase activity of tubulin-associated c-src.  One half of avian marrow macrophages maintained nonadherent  for 2 d were plated on tissue culture plastic while the other half  continued in suspension. After 90 min, the cells were lysed, and  the lysate was immunoprecipitated with antitubulin antibody.  One half the immunoprecipitate (A) was assayed for kinase activity by measuring 32P incorporation into c-src and enolase (an exogenous substrate). Tubulin content of the remaining immunoprecipitate was determined by immunoblot (B).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Tyrosine kinase activity of tubulin-associated c-src. One half of avian marrow macrophages maintained nonadherent for 2 d were plated on tissue culture plastic while the other half continued in suspension. After 90 min, the cells were lysed, and the lysate was immunoprecipitated with antitubulin antibody. One half the immunoprecipitate (A) was assayed for kinase activity by measuring 32P incorporation into c-src and enolase (an exogenous substrate). Tubulin content of the remaining immunoprecipitate was determined by immunoblot (B).
Mentions: To confirm the species immunoprecipitating with tubulin is, in fact, active c-src, we performed in vitro kinase assays on antitubulin immunoprecipitates, using as exogenous substrate, enolase, known to be specifically phosphorylated by the tyrosine kinase. Reflecting the abundance of c-src we find associated with tubulin in adherent osteoclast precursors, Fig. 7 A shows intense autophosphorylation of c-src and phosphorylation of exogenous substrate. In contrast to adherent cells, the magnitude of c-src autophosphorylation is much less pronounced in nonadherent macrophages (18% of the value in adherent cells normalized to protein levels), and phosphorylation of enolase is virtually undetectable. These differences in protein phosphorylation do not reflect altered tubulin abundance in adherent as compared to nonadherent cells (Fig. 7 B). The observed c-src kinase activity parallels, however, the amount of tubulin-associated c-src protein in adherent and nonadherent cells, respectively (Fig. 3 A).

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