Abstract
The two tandem homologous catalytic domains of PTPalpha possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTPalpha-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTPalpha-D1 and -D2 structures, identified two non-conserved amino acids in PTPalpha-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTPalpha-D1 positively affected the catalytic efficiency of PTPalpha-D2 toward the in vitro substrates para-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTPalpha-D2 into a phosphatase with catalytic efficiency for para-nitrophenylphosphate equal to PTPalpha-D1 but not approaching that of PTPalpha-D1 for the more complex substrate RR-src. In vivo, no gain in D2 activity toward p59(fyn) was effected by the double mutation. Alteration of the two corresponding invariant residues in PTPalpha-D1 to those in D2 conferred D2-like kinetics toward all substrates. Thus, these two amino acids are critical for interaction with phosphotyrosine but not sufficient to supply PTPalpha-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59(fyn). Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTPalpha remains an open question.
Highlights
Protein tyrosine phosphorylation status is a key determinant of most eukaryotic cell processes and is controlled by the protein-tyrosine kinases and phosphatases (PTPs).1 Phosphotyrosine hydrolysis is catalyzed by members of the large and diverse PTP superfamily, and the specific roles of most of these enzymes have yet to be determined, they can positively or negatively regulate cellular signaling pathways [1, 2]
The crystal structures of several PTPs support this mechanism and have further shown that the CX5R lies at the base of a phosphotyrosine-binding pocket, with substrate binding inducing the movement of a loop containing the WPD sequence so that the aspartate residue is brought into the catalytic site and in proximity to the leaving group oxygen [11,12,13]
The crystal structures of PTP1B complexed with phosphopeptide shows that the corresponding invariant tyrosine (Tyr-46) interacts with the phenyl ring of phosphotyrosine of the substrate [13]
Summary
Molecular Modeling—Modeling of structures was performed using LOOK (Molecular Applications Group). All mutations introduced by polymerase chain reaction were confirmed by DNA sequencing, and no extraneous mutations were found Restricted fragments of these mutants encompassing the desired mutations and most of the catalytic domain were used to replace homologous fragments of the full-length PTP␣ in the expression vector pXJ41-neo, where PTP␣ already contained an inactivating Cys to Ser mutation in D1 (C414S) and D2 (C704S) [30]. The Cys to Ser mutations are denoted in the figure legends as a subscript S following the domain containing the mutation These plasmids and those containing wild-type PTP␣ (pXJ41PTP␣-neo) [29] or PTP␣-D1(C414S)D2(C704S) [19] were subsequently used for cotransfection studies with p59fyn in COS-1 cells. Phosphatase Assays—The expression, purification, quantitation, and storage of GST-PTP␣ fusion proteins have been previously described [19]. The p59fyn level, phosphotyrosine content, and kinase activity were quantitated using a GS700 Bio-Rad densitometer
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