A Peptide that Stimulates Phosphorylation of the Plant Insulin‐Binding Protein

Abstract

The soybean seed basic 7 s globulin (Bg) is capable of binding bovine insulin and insulin-like growth factors, and has protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor. A 4-kDa peptide named leginsulin, which can bind to Bg and compete with insulin for binding to Bg, was isolated from radicles of germinated soybean seeds. The leginsulin had a stimulatory effect on the phosphorylation activity of Bg, suggesting that it is involved in cellular signal transduction. The leginsulin was sequenced by automated Edman degradation and electrospray ionization mass spectrometry. It consisted of 37 amino acid residues with six half-cystines in three disulfide bridges. The mass spectrometric analysis revealed that a portion of the peptide is processed to delete the C-terminal glycine like a number of animal peptide hormones, but not C-terminally amidated. The cDNA encoding the leginsulin was cloned, sequenced and considered to code for a precursor polypeptide consisting of a putative signal peptide, the leginsulin, a linker peptide, a 6-kDa peptide and a C-terminal peptide. Although there is no sequence similarity between the leginsulin and insulin or insulin-like growth factors, the leginsulin is a possible candidate for plant peptide hormones.The involvement of insulin-like polypeptides in regulatory metabolic mechanisms in plants and microorganisms is still under question. However, there is some information available on insulin-binding proteins within these organisms. In yeast, a plasma membrane protein was found to be capable of binding mammalian insulin [ l , 21. Recently, we have isolated a protein from soybean which bound insulin and insulin-like growth factor and which was found to be a seed basic 7 s globulin (Bg) [3,4]. In some other legume species and in carrot, related proteins were also shown to bind bovine insulin and insulin-like growth factors [4], suggesting that insulin-binding proteins are widely distributed in many plant species.Although amino acid sequence similarity was found in a limited region between Bg and the human insulin-binding protein [51], no sequence similarity was found between Bg and the human insulin-or insulin-like growth factor receptor [6, 71. However, Bg showed structural similarities to the insulin receptor in glycosylation, the presence of a cysteinerich domain and disulfide-bonded a and p (27 kDa and 16 kDa in Bg) subunits. Both proteins are also synthesized as larger precursor polypeptides which are post-translationally cleaved at the N-terminal side of a serine residue to generate a and j? subunits. In Bg, we found a consensus sequence for a nucleotide-binding site, indispensable for protein phosphorylation [8], and a protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor [9]. Furthermore, it was shown by immunocytochemistry that Bg is localized in the middle lamella of cell walls and the plasma membranes [lo]. These results suggest that Bg may...

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