Multiple Forms of Protein Kinases in Myelin and Microsomal Fractions of Bovine Brain

Saitoh

Yasugawa

et al. 1990

When the synaptosomal cytosol fraction from rat brain was chromatographed on a DEAE-cellulose column and assayed for protein phosphatases for tau factor and histone H1, two peaks of activities, termed peak 1 (major) and peak 2 (minor), were separated. Each peak was in a single form 2 (minor), were separated. Each peak was in a single form on Sephacryl S-300 column chromatography. Both peaks 1 and 2 dephosphorylated tau factor phosphorylated by Ca2+/calmodulin-dependent protein kinase II and the catalytic subunit of cyclic AMP-dependent protein kinase. The Km values were in the range of 0.42-0.84 microM for tau factor. There were no differences in kinetic properties of dephosphorylation between the substrates phosphorylated by the two kinases. The phosphatase activities did not depend on Ca2+, Mn2+ and Mg2+. Immunoprecipitation and immunoblotting analysis using polyclonal antibodies to the catalytic subunit of brain protein phosphatase 2A revealed that both protein phosphatases are the holoenzymic forms of protein phosphatase 2A. Aluminum chloride inhibited the activities of both peaks 1 and 2 with IC50 values of 40-60 microM. These results suggest that dephosphorylation of tau factor in presynaptic nerve terminals is controlled mainly by protein phosphatase 2A and that the neurotoxic effect of aluminum seems to be related mostly to inhibition of dephosphorylation of tau factor.

Section: Assay For Protein Phosphatasementioning

confidence: 99%

Dephosphorylation of r Factor by Protein Phosphatase 2A in Synaptosomal Cytosol Fractions, and Inhibition by Aluminum

Saitoh

Yasugawa

et al. 1990

“…The control sample was incubated under the same conditions except for the addition of boiled protein phosphatases. The reaction was terminated and the amount of phosphate incorporated into protein was measured as described previously by Miyamoto et al (1973Miyamoto et al ( , 1978.…”

Section: Assay For Dephosphorylation Of Proteinmentioning

confidence: 99%

Dephosphorylation of Microtubule Proteins by Brain Protein Phosphatases 1 and 2A, and Its Effect on Microtubule Assembly

Saitoh

Fukunaga

et al. 1988

114

Protein phosphatase C was purified 140-fold from bovine brain with 8% yield using histone H1 phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase (cyclic AMP-kinase). Brain protein phosphatase C was considered to consist of 10 and 90%, respectively, of the catalytic subunits of protein phosphatases 1 and 2A on the basis of the effects of ATP and inhibitor-2. Protein phosphatase C dephosphorylated microtubule-associated protein 2 (MAP2), tau factor, and tubulin phosphorylated by a multifunctional Ca2+/calmodulin-dependent protein kinase (calmodulin-kinase) and the catalytic subunit of cyclic AMP-kinase. The properties of dephosphorylation of MAP2, tau factor, and tubulin were compared. The Km values were in the ranges of 1.6-2.7 microM for MAP2 and tau factor. The Km value for tubulin decreased from 25 to 10-12.5 microM in the presence of 1.0 mM Mn2+. No difference in kinetic properties of dephosphorylation was observed between the substrates phosphorylated by the two kinases. Protein phosphatase C did not dephosphorylate the native tubulin, but universally dephosphorylated tubulin phosphorylated by the two kinases. The holoenzyme of protein phosphatase 2A from porcine brain could also dephosphorylate MAP2, tau factor, and tubulin phosphorylated by the two kinases. The phosphorylation of MAP2 and tau factor by calmodulin-kinase separately induced the inhibition of microtubule assembly, and the dephosphorylation by protein phosphatase C removed its inhibitory effect. These data suggest that brain protein phosphatases 1 and 2A are involved in the switch-off mechanism of both Ca2+/calmodulin-dependent and cyclic AMP-dependent regulation of microtubule formation.

“…Incubation was carried out at 30°C for 10 min in a shaking water bath. The reaction was terminated and the degree of protein phosphorylation measured as described previously (Miyamoto et al, 1973;Miyamoto et al, 1978).…”

mentioning

confidence: 99%

Purification and Characterization of a Ca²⁺‐ and Calmodulin‐Dependent Protein Kinase from Rat Brain

Fukunaga

Matsui

et al. 1982

209

A Ca2+- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited Km values of 109 and 30 microM for ATP and chicken gizzard myosin light chain, respectively, and Ka values of 12 nM and 1.9 microM for brain calmodulin and Ca2+, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a CA2+- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunits of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca2+- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.