Fatty acid-binding protein from rat heart is phosphorylated on Tyr19 in response to insulin stimulation.
The cytosolic fatty acid-binding protein from rat heart (H-FABP, M(r) 15,000) as well as FABP from mouse adipocytes (A-FABP, 62% homologous with H-FABP) contain a recognition sequence for protein tyrosine kinases, Asn-Phe-Asp-Asp-Tyr19. A-FABP has been shown by others to be partly phosphorylated on Tyr19, thus encouraging experiments designed to search for phosphotyrosine in H-FABP. For this purpose isolated cardiac myocytes were incubated with [32P]orthophosphate and analyzed by two-dimensional gel electrophoresis. A 15 kDa phosphoprotein present in the cytosolic protein fraction was specifically precipitated by a polyclonal antibody against rat heart FABP. Characterization of the phosphorylated FABP was facilitated by the development of an immunoaffinity purification procedure capable of isolating more than 200 micrograms FABP from four rat hearts in one step. Phosphoamino acid analysis and radiosequencing of the major tryptic phosphopeptide from immunopurified FABP revealed Tyr19 as the phosphorylated amino acid. Stimulation of cardiac myocytes with insulin in the presence of tyrosine phosphatase inhibitors led to a several-fold increase in the amount of phosphorylated FABP compared with a nearly undetectable level found without insulin stimulation, indicating that FABP may be a substrate for the insulin receptor tyrosine kinase. Phosphorylated FABP constitutes only a minor fraction compared to the large pool of FABP in the cardiac myocyte, thus obscuring the significance of this modification. However, as the phosphorylated Tyr19 residue is positioned within a helix-turn-helix-related domain of FABP, this modification may modulate a hitherto unknown DNA binding activity of FABP. A hypothesis is discussed in which phosphorylated FABP serves as a signalling molecule in the insulin signal transduction cascade.
Nielsen
S U
SU
Department of Biochemistry, University of Münster, Germany.
Spener
F
F
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D005260:Female; D018257:Helix-Loop-Helix Motifs; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis; D009206:Myocardium / Q000378:metabolism*; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D002352:Carrier Proteins; C490174:Fabp7 protein, rat; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D007328:Insulin; D009124:Muscle Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases