Kl. Hippen et al., ACIDIC RESIDUES ARE INVOLVED IN SUBSTRATE RECOGNITION BY 2 SOLUBLE-PROTEIN TYROSINE PHOSPHATASES, PTP-5 AND RRBPTP-1, Biochemistry, 32(46), 1993, pp. 12405-12412
The mechanisms for substrate recognition by two cytoplasmic protein ty
rosine phosphatases, PTP-5 and rrbPTP-1, were investigated. Phosphoryl
ation sites on tyrosine-phosphorylated casein, a model PTP substrate,
were characterized. Two peptides based on casein phosphorylation sites
and one peptide based on the tyrosine phosphorylation site of reduced
, carboxamidomethylated and maleylated (RCM) lysozyme were tested as P
TP substrates. The three peptides were dephosphorylated by PTP-5 and r
rbPTP-1 at rates comparable to those of the corresponding sites on the
intact proteins. This indicates that peptides based on the two model
PTP substrates, casein and RCM-lysozyme, contained all or most of the
structural information necessary for PTP-5 and rrbPTP-1 substrate reco
gnition. Structural elements required for substrate recognition by PTP
-5 and rrbPTP-1 were also investigated. K(m) values for dephosphorylat
ion of three simple aromatic phosphate esters (phosphotyrosine, p-nitr
ophenyl phosphate, and phenyl phosphate) by rrbPTP-1 were about 5000-f
old higher than those obtained for the peptide and protein substrates.
This indicates that recognition of protein and peptide substrates inv
olves structural elements in addition to the phosphate group and the a
romatic tyrosine ring of phosphotyrosine. Analysis of the effects of t
runcations and Ala for polar substitutions on the reactivity with PTP-
5 and rrbPTP-1 of peptides based on casein, RCM-lysozyme, and angioten
sin II indicated that Asp or Glu within the first five residues on the
N-terminal side of phosphotyrosine increased peptide reactivity with
both PTP's. Asn residues were unable or only weakly able to substitute
for Asp residues. These results indicate that one or more acidic resi
dues on the N-terminal side of phosphotyrosine enhance peptide reactiv
ity with PTP-5 and rrbPTP-1 in an additive fashion.