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Autophosphorylation-Dependent Activation of a
Calcium-Dependent Protein Kinase from Groundnut1
Subho Chaudhuri,
Anindita Seal, and
Maitrayee Das Gupta*
Department of Biochemistry, Ballygunge Science College, Calcutta
University, 35 Ballygunge Circular Road, Calcutta 700019, India
Ca2+-dependent protein
kinases (CDPKs) containing a calmodulin-like domain integrated in
their primary sequence are present primarily in plants. A member of
this family was characterized from the groundnut (Arachis
hypogea) plant and called GnCDPK (M. DasGupta [1994]
Plant Physiol 104: 961-969). GnCDPK specifically uses the
myosin light chain synthetic peptide (MLCpep), which is the
phosphate-accepting domain of smooth muscle myosin light chains
(KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental
conditions. In this report we show that GnCDPK undergoes intramolecular
autophosphorylation. This self-phosphorylation occurs in threonine
residues in a Ca2+-dependent
(K0.5 = 0.5 µM) and
calmodulin-independent manner. The kinase activity toward MLCpep and
its sensitivity to Ca2+ were unaffected by prior
autophosphorylation when measured under saturating ATP concentrations.
The role of autophosphorylation in the exogenous substrate MLCpep
phosphorylation reaction was reinvestigated at low ATP concentrations.
A pronounced lag time of 1 to 2 min, followed by a linear increase of
activity for 7.5 min, was seen in the initial rate of MLCpep
phosphorylation under such suboptimal conditions. Prior
autophosphorylation completely abolished this lag phase, and a sharp
rise of exogenous substrate phosphorylation was seen from the 1st min.
Our results suggest that autophosphorylation is a prerequisite for the
activation of GnCDPK.
1
This work was supported by the Council of
Scientific and Industrial Research (grant no. 38-0894-95-EMR II),
Government of India.
*
Corresponding author; e-mail maitrayee_d{at}hotmail.com; fax
91-33-476-4419.
Plant Physiol. (1999) 120: 859-866
Copyright Clearance Center: 0032-0889/99/120//08
© 1999 American Society of Plant Physiologists
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