Detection of a Calcium-Activated Protein Kinase in Mougeotia by Using Synthetic Peptide Substrates

Abstract

By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulindependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V.I/Km ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity.

Analysis of the State of Posttranslational Calmodulin Methylation in Developing Pea Plants

“…Radiometric calmodulin N-methyltransferase assay procedures were done as previously described (25 dependence of the enzyme, the incubation buffer was modified to contain 25 mm Hepes NaOH (pH 7.5), 2 mm DTT, containing 0.01% (w/v) Triton X-100, and various amounts of MgCl2, CaCl2, and EGTA designed to give a Mg2+ concentration of 5 mm and a free Ca2+ concentration that varied from 10-2 to 10-9 M. Chelex treatment of buffers and quantitation of metal ions by atomic absorption spectroscopy were done as previously described (24).…”

Peptide phosphorylation by calcium‐dependent protein kinase from maize seedlings

“…Fractions from the Mono Q column with CDPK activity were incubated with 0.1 mm ATP labelled with [g-32 P]ATP (specific radioactivity < 100 c.p.m.´pmol 21 ) and 5 mm MgCl 2 at 25 8C for 30 min. The reaction was terminated by dilution of the incubation mixture 1.5 times with SDS/PAGE sample buffer containing 65 mm Tris/HCl, pH 6.8, 10% glycerol, 2.1% SDS and 5% 2-mercaptoethanol.…”

“…For each peptide, the reaction time and enzyme amount were adjusted so that the initial rates of the phosphorylation reaction could be measured. The reactions were initiated by addition of the [g-32 P]ATP (specific radioactivity 50±150 c.p.m.´pmol 21 ) and monitored by the phosphocellulose paper method as described above. It was confirmed by the method used in [13] that the difference in radioactivity for different peptides was not a consequence of deviations in their binding to the phosphocellulose paper.…”

“…The specific radioactivity of [g-32 P]ATP was 50±150 c.p.m.´pmol 21 . The phosphorylation reactions were initiated by immersing the SPOTs membrane in the incubation mixture, using 100±150 mL of solution for each peptide spot.…”

“…Fractions of volume 50 mL were collected and pooled according to the peak of protein kinase activity. A typical phosphorylation assay for determination of protein kinase activity consisted of 10 mL sample fraction, 10 mL 150 mm Tris/HCl/5 mm CaCl 2 , pH 7.5, 10 mL 20 mm Tris/HCl, pH 7.5 (containing lipids when tested), 20 mL 3.75 mg´mL 21 histone or 100 mm peptide VLARLHSVRER or LLRLHSLRE and 10 mL 0.75 mm [g-32 P]ATP/37.5 mm MgCl 2 . When the stimulation of the protein kinase activity by phospholipid and diacylglycerol was tested, 60 mg´mL 21 phosphatidylserine and 1 mg´mL 21 diolein were included in the mixture.…”