Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase α

Abstract

cDNA expression library screening revealed binding between the membrane distal catalytic domain (D2) of protein-tyrosine phosphatase α (PTPα) and calmodulin. Characterization using surface plasmon resonance showed that calmodulin bound to PTPα-D2 in a Ca2+-dependent manner but did not bind to the membrane proximal catalytic domain (D1) of PTPα, to the two tandem catalytic domains (D1D2) of PTPα, nor to the closely related D2 domain of PTPε. Calmodulin bound to PTPα-D2 with high affinity, exhibiting a K(D) ~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 PTPε sequence could confer calmodulin binding. The overlap of the binding site with the predicted lip of the catalytic cleft of PTPα-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (K(i) ~340 nM), suggests that binding of calmodulin physically blocks or distorts the catalytic cleft of PTPα-D2 to prevent interaction with substrate. When expressed in cells, full-length PTPα and PTPα lacking only D1, but not full-length PTPε, bound to calmodulin beads in the presence of Ca2+. Also, PTPα was found in association with calmodulin immunoprecipitated from cell lysates. Thus calmodulin does associate with PTPα in vivo but not with PTPα-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 PTPα-D2 and, via D2, of PTPα.