Calcium-stimulated Autophosphorylation Site of Plant Chimeric Calcium/Calmodulin-dependent Protein Kinase
Journal of Biological Chemistry
The existence of two molecular switches regulating plant chimeric Ca 2؉ /calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca 2؉ -sensitive molecular switch and calmodulin binding domain acting as Ca 2؉ -stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca 2؉ -stimulated
... ted autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr 267 was confirmed as the Ca 2؉ -stimulated autophosphorylation site by post-source decay experiments and by sitedirected mutagenesis. The purified T267A mutant form of CCaMK did not show Ca 2؉ -stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca 2؉ /calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca 2؉ messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca 2؉ -stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca 2؉ -stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca 2؉ -stimulated phosphorylation occurs by an intermolecular mechanism. Intracellular calcium signals commonly exert their effects through the regulation of protein phosphorylation (2-5). These signaling events are orchestrated by calcium-binding proteins that decode information contained in the spatial and temporal patterns of the Ca 2ϩ signals. In addition to calmodulin, a major transducer of Ca 2ϩ signals, plants contain Ca 2ϩ /calmodulin (CaM) 1 -dependent protein kinases and a large family of Ca 2ϩ -dependent but CaM-independent protein kinases (CDPKs) and CDPK-related protein kinases. Ca 2ϩ -dependent protein phosphorylation and Ca 2ϩ /CaM-dependent protein phosphorylation are believed to participate in numerous aspects of plant growth and development (2, 6). Several Ca 2ϩ /CaM-dependent protein kinases have been known to exist in animal systems, and numerous reports have appeared in the literature about their characterization and functional significance. However, not much is known about the Ca 2ϩ /CaM-dependent protein kinases in plants (7-10). The chimeric Ca 2ϩ /CaM-dependent protein kinase (CCaMK) reported from plants has a C-terminal visinin-like domain (9, 10) unlike all the other Ca 2ϩ /CaM-dependent protein kinases reported so far. CCaMK is expressed in a tissue-specific manner, and it is developmentally regulated (11). Apart from the C-terminal visinin-like domain, CCaMK has a serine-threonine kinase domain and an autoinhibitory domain overlapping with the CaM binding domain. The CaM binding domain of CCaMK is highly similar to mammalian Ca 2ϩ /CaM-dependent protein kinase II (12). Although there are several reports on Ca 2ϩ and Ca 2ϩ /CaM-dependent protein phosphorylation in plants and their role in plant growth and development, the precise molecular nature of Ca 2ϩ and Ca 2ϩ / CaM-dependent protein phosphorylation is not well understood. In the present study, we identify and characterize the Ca 2ϩstimulated autophosphorylation site of CCaMK by MALDI-TOF mass spectrometry and PSD experiments. The threonine 267 was found to be phosphorylated upon Ca 2ϩ stimulation, and the T267A mutant did not show Ca 2ϩ -stimulated autophosphorylation. Furthermore, Ca 2ϩ /CaM-stimulated kinase activity and autophosphorylation-dependent CaM affinity changes were absent in the mutant. Identification of Thr 267 as the Ca 2ϩ -stimulated autophosphorylation site of CCaMK is an important step in the understanding of the molecular nature of transduction of Ca 2ϩ signals into phosphorylation signals in plants. We show that a synthetic peptide (amino acid residues 257-340) containing the autophosphorylation site and CaMbinding site could kinetically mimic the high affinity (phosphorylated) and low affinity (unphosphorylated) binding of CCaMK to CaM. EXPERIMENTAL PROCEDURES Expression and Purification of CCaMK-CCaMK cDNA from Lily (Lilium longiflorum Thunb cv. Nellie white) and deletion mutants were cloned into the pET3b expression vector (Novagen) and expressed in Escherichia coli. These proteins were purified as described previously (12).