Phosphorylated residues as specificity determinants for an acidophilic protein tyrosine kinase: A study with src and cdc2 derived phosphopeptides

• Published in: FEBS letters Vol. 330; no. 2; pp. 141 - 145
• Main Authors: Donella-Deana, Arianna, Marin, Oriano, Brunati, Anna Maria, Cesaro, Luca, Piutti, Claudia, Pinna, Lorenzo A.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 13-09-1993; Elsevier
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; CDC2 Protein Kinase - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hierarchal phosphorylation; Kinetics; Molecular Sequence Data; Oligopeptides - chemistry; Oligopeptides - metabolism; Phosphopeptide; Phosphoproteins - metabolism; Phosphorylation; Protein tyrosine kinase; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins pp60(c-src) - metabolism; Rats; Specificity determinant; Spleen; Spleen - enzymology; Substrate Specificity; Transferases; Spleen; Hierarchal phosphorylation; Phosphopeptide; PKA, cAMP-dependent protein kinase; MCPBA, m-chloroperoxybenzoic acid; DCM, dichloromethane; RP-HPLC, reverse-phase high-performance liquid chromatography; GSK3, glycogen synthase kinase 3; CK1, casein kinase 1; Specificity determinant; Protein tyrosine kinase; TBTU, 2-benzotriazolyl-tetramethyluronium tetrafluoroborate; PMSF, phenylmethylsulphonyl fluoride; Fmoc, fluorenylmethoxycarbonyl; HOBt. 1-hydroxybenzotriazole; DMF, NN-dimethylformamide; CK2, casein kinase 2; Site specificity; Vertebrata; Phosphorylation; Mammalia; Enzymatic activity; Rat; Enzyme; Transferases; Substrate specificity; Rodentia; Protein-tyrosine kinase
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80260-2

Spleen TPK-IIB is an acidophilic protein tyrosine kinase, devoid of autophosphorylation activity, whose phosphorylation of the
src-peptide NEYTA is crucially specified by Glu-2 [(1991) J. Biol. Chem. 266,17798-17803]. We show that phosphothreonine, phosphotyrosine and phosphoserine are, in this order, specificity determinants even more effective than glutamic acid if they are replacing Glu-2, to give the phosphopeptides NTpYTA, NYpYTA, NSpYTA, respectively. Non-phosphorylated threonine, tyrosine and serine are conversely ineffective. Consequently also the heptapeptide GEGTYGV reproducing the phosphoacceptor and inhibitory site of p34
cdc2 is not appreciably affected by TPK-IIB, unless its threonyl residue is previously phosphorylated, the phosphoderivative GEGTpYGV being readily phosphorylated at its tyrosyl residue. Such a behaviour is unique for TPK-IIB among the protein tyrosine kinases tested (
lyn-TPK,
fgr-TPK and EGF-receptor, besides TPK-IIB). These data provide the first evidence that, in some instances, the targeting by protein tyrosine kinases can be specifically determined by the previous phosphorylation of the peptide substrate, thus extending the concept of ‘hierarchal phosphorylation’ [(1991) J. Biol. Chem. 266, 14139-14142] to tyrosyl residues as well.