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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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Subcellular colocalization of vacuolar H+-ATPase and  c-src. Cells maintained on tissue culture plates for 3 d were lysed  (S0), and the lysate was centrifuged at 1,000 g to remove nuclei  and large cell debris. Supernatant was centrifuged at 27,000 g and  the membrane pellet (P3) was fractionated on a sucrose density  gradient. An equal amount of protein from each gradient fraction  was subjected to immunoblot using antibodies to c-src, H+-ATPase, and F4/80. GLF, Golgi light; GHF, Golgi heavy; CVCF,  crude vesicle fractions.
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Figure 16: Subcellular colocalization of vacuolar H+-ATPase and c-src. Cells maintained on tissue culture plates for 3 d were lysed (S0), and the lysate was centrifuged at 1,000 g to remove nuclei and large cell debris. Supernatant was centrifuged at 27,000 g and the membrane pellet (P3) was fractionated on a sucrose density gradient. An equal amount of protein from each gradient fraction was subjected to immunoblot using antibodies to c-src, H+-ATPase, and F4/80. GLF, Golgi light; GHF, Golgi heavy; CVCF, crude vesicle fractions.

Mentions: To determine if the osteoclast H+-ATPase and c-src colocalize, we analyzed density gradient–separated membrane fractions derived from generated avian osteoclasts. Fig. 16 shows that in contrast to the Golgi heavy fraction, which contains H+-ATPase but not c-src, both molecules are present in the Golgi light fraction. Documenting specificity, F4/80, a cell surface marker of mature macrophages, is present in total cell lysate but not in intracellular fractions.


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)

Subcellular colocalization of vacuolar H+-ATPase and  c-src. Cells maintained on tissue culture plates for 3 d were lysed  (S0), and the lysate was centrifuged at 1,000 g to remove nuclei  and large cell debris. Supernatant was centrifuged at 27,000 g and  the membrane pellet (P3) was fractionated on a sucrose density  gradient. An equal amount of protein from each gradient fraction  was subjected to immunoblot using antibodies to c-src, H+-ATPase, and F4/80. GLF, Golgi light; GHF, Golgi heavy; CVCF,  crude vesicle fractions.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 16: Subcellular colocalization of vacuolar H+-ATPase and c-src. Cells maintained on tissue culture plates for 3 d were lysed (S0), and the lysate was centrifuged at 1,000 g to remove nuclei and large cell debris. Supernatant was centrifuged at 27,000 g and the membrane pellet (P3) was fractionated on a sucrose density gradient. An equal amount of protein from each gradient fraction was subjected to immunoblot using antibodies to c-src, H+-ATPase, and F4/80. GLF, Golgi light; GHF, Golgi heavy; CVCF, crude vesicle fractions.
Mentions: To determine if the osteoclast H+-ATPase and c-src colocalize, we analyzed density gradient–separated membrane fractions derived from generated avian osteoclasts. Fig. 16 shows that in contrast to the Golgi heavy fraction, which contains H+-ATPase but not c-src, both molecules are present in the Golgi light fraction. Documenting specificity, F4/80, a cell surface marker of mature macrophages, is present in total cell lysate but not in intracellular fractions.

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