PLANT PHYSIOLOGY , Vol 103, Issue 3 955-962, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro
S. Zhang, C. D. Jin and S. J. Roux
Department of Botany, The University of Texas at Austin, Austin, Texas 78713-7640
A casein kinase II-type protein kinase has been purified from the cytosolic
fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity
as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide
gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a
native molecular mass of 150 kD, and subunit molecular masses of 41 and 40
kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the
putative regulatory subunit ([beta]).Casein and phosvitin can be used as
artificial substrates for this kinase. Both serine and threonine residues
were phosphorylated when mixed casein, [beta]-casein, or phosvitin were
used as the substrate, whereas only serine was phosphorylated if
[alpha]-casein or histone III-S was the substrate. The kinase activity was
stimulated 130% by 0.5 mM spermine (the concentration required for 50% of
maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 =
0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was
very sensitive to inhibition by heparin (concentration for 50% inhibition
[I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type
protein kinases, this preparation was inhibited by K+ and Na+, with I50
values of 75 and 65 mM, respectively. Pretreatment of the purified kinase
preparation in vitro with alkaline phosphatase caused a 5-fold decrease in
its activity. Additionally, this kinase also lost its activity when its
[beta] subunit was autophosphorylated in the absence of substrate. These
results suggest that the activity of this casein kinase II protein kinase
may be regulated by the phosphorylation state of two different sites in its
multimeric structure.
