Expression and characterization of wild type, truncated, and mutant forms of the intracellular region of the receptor-like protein tyrosine phosphatase HPTP beta.
Human HPTP beta is unique among mammalian receptor-like protein tyrosine phosphatases in that it has only a single catalytic domain. The intracellular region of HPTP beta was expressed in bacteria, purified, and characterized. It exhibits high activity toward all substrates tested and is potently inhibited by zinc. Vanadate and polyanions also inhibited activity. The juxta-membrane segment of HPTP beta (residues 1622-1639) potentially functions as a negative regulatory sequence since its deletion can increase HPTP beta activity 5-fold. This segment contains up to two sites for protein kinase C phosphorylation, although in vitro phosphorylation by this kinase did not affect HPTP beta activity. The boundaries of the catalytic domain were delineated by truncation analyses. Successive deletion of N-terminal sequence prior to residue 1684 had little effect on substrate affinity and at most reduced activity about 6-fold. Further removal of residues 1684-1686 resulted in a marked 50-500-fold drop in activity, and loss of N-terminal sequence prior to residue 1690 abolished activity. Based on these analyses a highly conserved motif was identified in all mammalian tyrosine phosphatases (E/q) (F/y)XX(L/i), corresponding to positions 1684-1688 of HPTP beta. Mutation of residue 1684 or 1685 generally gave rise to proteins with marked temperature sensitivity. These mutant HPTP beta were active but had reduced activity compared to the wild type enzyme. In conjunction, these results suggest that this region represents the N-terminal border of the catalytic domain and is essential for correct phosphatase folding although not directly involved in catalysis. Parallel truncation studies have defined residues 1930-1939/40 as the C-terminal border of the catalytic domain.
Wang
Y
Y
Cell Regulation Laboratory, National University of Singapore, Kent Ridge, Crescent.
Pallen
C J
CJ
D000595:Amino Acid Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013816:Thermodynamics
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D011493:Protein Kinase C; D017027:Protein Tyrosine Phosphatases