Src homology-2 domains protect phosphotyrosyl residues against enzymatic dephosphorylation.
The SH2 domain of c-Fgr (class 1A) has been expressed in E. coli as GST fusion protein and tested for its ability to prevent the dephosphorylation of a variety of phosphotyrosyl (poly)peptides by three distinct protein tyrosine phosphatases (TC-PTPase, YOP, and Low Mr PTPase). Dephosphorylation of HS1 protein and of a derived phosphopeptide, HS1 (388-402), exhibiting the motif selected by class 1A SH2 domains is inhibited in a dose dependent manner with full inhibition promoted by a 2- to 3-molar excess of GST/SH2 domain irrespective of either the nature or the amount of phosphatase used. The IC50 values for inhibition of these and other phosphotyrosyl substrates roughly correlates with their expected affinity for class 1A SH2 domain. Inhibition is partially reversed by the addition of D-myo-inositol 1,4,5-triphosphate, which competes for the binding to the SH2 domains. Our data on one side show that additional mechanism(s) besides mere competition must assist PTPases to dissociate SH2-PTyr complexes and on the other suggest a role for SH2 domains in protecting phosphotyrosyl residues from premature dephosphorylation.
Brunati
A M
AM
Dipartimento di Chimica Biologica, Universita' di Padova, Italy.
Pinna
L A
LA
Bergantino
E
E
Ruzzene
M
M
Cirri
P
P
Ramponi
G
G
Donella-Deana
A
A
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D018909:src Homology Domains; D019061:src-Family Kinases / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D004791:Enzyme Inhibitors; C061909:HCLS1 protein, human; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D015544:Inositol 1,4,5-Trisphosphate; D005982:Glutathione Transferase; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases; D017027:Protein Tyrosine Phosphatases