SUBSTRATE RECOGNITION BY OSTEOCLAST PRECURSORS INDUCES C-SRC MICROTUBULE ASSOCIATION/

Citation
Y. Abuamer et al., SUBSTRATE RECOGNITION BY OSTEOCLAST PRECURSORS INDUCES C-SRC MICROTUBULE ASSOCIATION/, The Journal of cell biology, 137(1), 1997, pp. 247-258
Citations number
55
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
00219525
Volume
137
Issue
1
Year of publication
1997
Pages
247 - 258
Database
ISI
SICI code
0021-9525(1997)137:1<247:SRBOPI>2.0.ZU;2-X
Abstract
The osteoclast is distinguished from other macrophage polykaryons by i ts 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 bon e apposed plasmalemma, The failure of osteoclasts in c-src-/- osteopet rotic 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 c ase, 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 p recursors) maintained in the adherent, but not nonadherent, state. Sin ce the two proteins colocalize only within adherent avian osteoclast-l ike 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 recogni tion, and the event does not reflect increased expression of either pr otein. In vitro kinase assay demonstrates tubulin-associated c-src is enzymatically active, phosphorylating itself as well as exogenous subs trate. The increase in microtubule-associated kinase activity attendin g adhesion mirrors tubulin-bound c-src and does not reflect enhanced s pecific activity. The fact that microtubule-dissociating drugs, as wel l as cold, prevent adherence-induced c-src/tubulin association indicat es the protooncogene complexes primarily, if not exclusively, with pol ymerized 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. Finall y, consistent with cotransport of c-src and the osteoclast vacuolar pr oton pump to the polarized plasmalemma, the H+-ATPase decorates microt ubules in a manner similar to the protooncogene, specifically coimmuno precipitates with c-src from the osteoclast light Golgi membrane fract ion, and is present, with c-src, in preparations enriched with acidify ing vesicles reconstituted from the osteoclast ruffled membrane.