Site specificity of p72syk protein tyrosine kinase: efficient phosphorylation of motifs recognized by Src homology 2 domains of the Src family.
Protein tyrosine kinase p72syk purified from rat spleen has been assayed for its ability to phosphorylate a number of peptide substrates derived from naturally occurring phospho-acceptor sites. The phosphorylation efficiency is extremely variable, depending on the peptide sequence, with Km values in the 3-1500 microM range. The by far best peptide substrates, with Km values of 3 and 4 microM are those reproducing the phospho-acceptor sites of Vav and HS1 proteins, respectively. These sites include multiple acidic residues flanking tyrosine on both sides and they also display the consensus sequences (YEDL and YEEV) preferred by the SH2 domains of the Src family. Alteration of this consensus in the HS1 peptide, by replacing either the glutamic acid or valine, also reduces the phosphorylation efficiency by p72syk. Also the replacement of acidic residues at position -1 and, to a lesser extent at positions -3 and -4 (but not at positions +3 and +5) are detrimental. These observations may suggest a role of p72syk in the recruitment of ligands/substrates for the Src family enzymes. We also show that the HS1 peptide can be used for the specific monitoring of p72syk since neither the two Src-related c-Fgr and Lyn kinases (needing a hydrophobic instead of acidic residue at position -1) nor CSK appreciably phosphorylate it.
Brunati
A M
AM
Dipartimento di Chimica Biologica, Università di Padova, Italy.
Donella-Deana
A
A
Ruzzene
M
M
Marin
O
O
Pinna
L A
LA
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity; D000072377:Syk Kinase
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D004792:Enzyme Precursors; C061909:HCLS1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; C040475:Src peptide; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606898:Syk protein, rat