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.
Highlights
Protein-tyrosine phosphatase ␣ (PTP␣)1 is a ubiquitously expressed, yet brain-enriched, receptor that acts as a positive regulator of the tyrosine kinases Src and Fyn [1,2,3,4,5], as a regulator of the Kv1.2 potassium channel [6], and as a potential modulator of insulin receptor signaling [7]
PTP␣-D2 bound in a Ca2ϩ-dependent manner to bovine brain calmodulin coated on the sensor chip, with no binding observed in the presence of EGTA (Fig. 1A)
Increasing concentrations of PTP␣-D2 resulted in increased binding (RU) to the calmodulin-coated sensor chip
Summary
Expression Plasmids—Numbering of the PTP␣ and PTP⑀ amino acid sequences is according to Krueger et al [32]. The plasmid pGEX-4T1-PTP␣-D2C was constructed by insertion of a D2 fragment corresponding to aa 543–774 into pGEX-4T1 (Amersham Pharmacia Biotech). The primer sequences were 5ЈGGGGATCCAACAATGGATTAGAGGAG-3Ј and 5Ј-GGCCGGGATCTTGTCATTCTGGAT-3Ј for PTP␣-N1, 5Ј-GGGGATCCGACAAGATGCGGACTGGA-3Ј and 5Ј-GGGAATTCCTGTAAAACACGGTTCTT-3Ј for PTP␣-N2, and 5Ј-GGGGATCCGAGAACATGAGGACGGGC-3Ј and 5ЈGGGAATTCCTGGATGACCCTGGCCTT-3Ј for PTP⑀-N2 These polymerase chain reaction fragments were subcloned in-frame into pGEX-4T1-PTP␣-D2C. The immunoprecipitates were washed five times in buffer B (20 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mM CaCl2), resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted using the ECL system (Amersham Pharmacia Biotech). In Vitro Binding Assay Using Calmodulin-Sepharose Beads—Lysates of COS-1 cells expressing VSVG-PTP␣, VSVG-PTP␣-D2⌬D1, or FLAG-PTP⑀ were incubated with 50 l of calmodulin-Sepharose beads (Amersham Pharmacia Biotech) in the presence of 1 mM of CaCl2 or EGTA at 4 °C for 3 h. The eluates were resolved by SDS-PAGE and immunoblotted with anti-VSVG or anti-FLAG antibody
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