title: The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity.
abstract: Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.
authors.affiliation: Centre for Diabetes and Endocrine Research, Princess Alexandra Hospital, University of Queensland, Brisbane, Qld 4102, Australia.
authors.forename: Elaine C
authors.initials: EC
authors.lastname: Thomas
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhe
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Molero
authors.forename: Carsten
authors.initials: C
authors.lastname: Schmitz-Peiffer
authors.forename: Georg
authors.initials: G
authors.lastname: Ramm
authors.forename: David E
authors.initials: DE
authors.lastname: James
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Whitehead
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000737:chemistry* / Q000648:ultrastructure*; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D014404:Tubulin / Q000378:metabolism; D014746:Vimentin / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D014404:Tubulin; D014746:Vimentin; D051057:Proto-Oncogene Proteins c-akt