title: Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase alpha.
abstract: cDNA expression library screening revealed binding between the membrane distal catalytic domain (D2) of protein-tyrosine phosphatase alpha (PTPalpha) and calmodulin. Characterization using surface plasmon resonance showed that calmodulin bound to PTPalpha-D2 in a Ca(2+)-dependent manner but did not bind to the membrane proximal catalytic domain (D1) of PTPalpha, to the two tandem catalytic domains (D1D2) of PTPalpha, nor to the closely related D2 domain of PTPepsilon. Calmodulin bound to PTPalpha-D2 with high affinity, exhibiting a K(D) approximately 3 nm. The calmodulin-binding site was localized to amino acids 520-538 in the N-terminal region of D2. Site-directed mutagenesis showed that Lys-521 and Asn-534 were required for optimum calmodulin binding and that restoration of these amino acids to the counterpart PTPepsilon sequence could confer calmodulin binding. The overlap of the binding site with the predicted lip of the catalytic cleft of PTPalpha-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (K(i) approximately 340 nm), suggests that binding of calmodulin physically blocks or distorts the catalytic cleft of PTPalpha-D2 to prevent interaction with substrate. When expressed in cells, full-length PTPalpha and PTPalpha lacking only D1, but not full-length PTPepsilon, bound to calmodulin beads in the presence of Ca(2+). Also, PTPalpha was found in association with calmodulin immunoprecipitated from cell lysates. Thus calmodulin does associate with PTPalpha in vivo but not with PTPalpha-D1D2 in vitro, highlighting a potential conformational difference between these forms of the tandem catalytic domains. The above findings suggest that calmodulin is a possible specific modulator of PTPalpha-D2 and, via D2, of PTPalpha.
authors.affiliation: Cell Regulation Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Singapore.
authors.forename: L
authors.initials: L
authors.lastname: Liang
authors.forename: K L
authors.initials: KL
authors.lastname: Lim
authors.forename: K T
authors.initials: KT
authors.lastname: Seow
authors.forename: C H
authors.initials: CH
authors.lastname: Ng
authors.forename: C J
authors.initials: CJ
authors.lastname: Pallen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance
chemical_list: D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D017027:Protein Tyrosine Phosphatases