Interconversion of the Kinetic Identities of the Tandem Catalytic Domains of Receptor-like Protein-tyrosine Phosphatase PTPα by Two Point Mutations Is Synergistic and Substrate-dependent*
- From the Cell Regulation Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609 and the‡Bioinformatics Center, National University of Singapore, 5 Lower Kent Ridge Road, National University Hospital, Singapore 119074, Republic of Singapore
Abstract
The two tandem homologous catalytic domains of PTPα possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTPα-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTPα-D1 and -D2 structures, identified two non-conserved amino acids in PTPα-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTPα-D1 positively affected the catalytic efficiency of PTPα-D2 toward the in vitro substratespara-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTPα-D2 into a phosphatase with catalytic efficiency forpara-nitrophenylphosphate equal to PTPα-D1 but not approaching that of PTPα-D1 for the more complex substrate RR-src.In vivo, no gain in D2 activity toward p59fyn was effected by the double mutation. Alteration of the two corresponding invariant residues in PTPα-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 PTPα-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59fyn. Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTPα remains an open question.
Footnotes
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↵* This work was supported by the National Science and Technology Board of Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed. Tel.: 65-874-3742; Fax: 65-779-1117; E-mail: mcbcp{at}imcb.nus.edu.sg.
- Abbreviations:
- PTP
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protein-tyrosine phosphatase
- GST
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glutathioneS-transferase
- pNPP
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para-nitrophenylphosphate
- RPTP
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receptor protein-tyrosine phosphatase
- Pipes
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1,4-piperazinediethanesulfonic acid.
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- Received June 10, 1998.
- Revision received August 6, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
