Calmodulin Binds to and Inhibits the Activity of the Membrane
Distal Catalytic Domain of Receptor Protein-tyrosine Phosphatase
*
Lu
Liang
,
Kah Leong
Lim,
Kah Tong
Seow§,
Chee Hoe
Ng, and
Catherine J.
Pallen¶
From the Cell Regulation Laboratory, Institute of
Molecular and Cell Biology, 30 Medical Drive,
Singapore 117609, Singapore
cDNA expression library screening revealed
binding between the membrane distal catalytic domain (D2) of
protein-tyrosine phosphatase 
(PTP) and calmodulin.
Characterization using surface plasmon resonance showed that calmodulin
bound to PTP-D2 in a Ca2+-dependent
manner but did not bind to the membrane proximal catalytic domain (D1)
of PTP, to the two tandem catalytic domains (D1D2) of PTP, nor to
the closely related D2 domain of PTP
. Calmodulin bound to PTP-D2
with high affinity, exhibiting a KD ~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 PTP sequence could confer calmodulin binding. The
overlap of the binding site with the predicted lip of the catalytic
cleft of PTP-D2, in conjunction with the observation that calmodulin acts as a competitive inhibitor of D2-catalyzed dephosphorylation (Ki ~340 nM), suggests that binding
of calmodulin physically blocks or distorts the catalytic cleft of
PTP-D2 to prevent interaction with substrate. When expressed in
cells, full-length PTP and PTP lacking only D1, but not
full-length PTP, bound to calmodulin beads in the presence of
Ca2+. Also, PTP was found in association with calmodulin
immunoprecipitated from cell lysates. Thus calmodulin does associate
with PTP in vivo but not with PTP-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 PTP-D2 and, via
D2, of PTP.
*
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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