Insulin and insulin-like growth factor I (IGF I) stimulate phosphorylation of a Mr 175,000 cytoskeleton-associated protein in intact FRTL5 cells.
FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells.
Condorelli
G
G
Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Naples, Italy.
Formisano
P
P
Villone
G
G
Smith
R J
RJ
Beguinot
F
F
D000818:Animals; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000494:pharmacology*; D013961:Thyroid Gland
D003598:Cytoskeletal Proteins; D007328:Insulin; D010750:Phosphoproteins; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin