Hitoshi Fujisawa

A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel.

A procedure for detecting protein kinase activities of the alpha and beta subunits of calmodulin-dependent protein kinase II separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. After electrophoresis, the gel was immersed in 6 M guanidine HCl for 1 h and then in a buffer containing 0.04% Tween 40 for 16 h at 4 degrees C for renaturation of the resolved polypeptides. The renatured polypeptides in the gel were incubated with [gamma-32P]ATP for phosphorylation of either the substrate included in the polyacrylamide gel or the kinase itself. After removal of the unreacted [gamma-32P]ATP, the protein kinase activities were visualized by autoradiography. Two radioactive protein bands of Mr 50,000 and 60,000, which corresponded to the alpha and beta subunits, were detected only when the phosphorylation was carried out in the presence of Ca2+ and calmodulin. Approximately 0.05 micrograms of the enzyme could be detected on a gel containing no protein substrate. When microtubule-associated protein 2 was included in the gel, the sensitivity of the detection of calmodulin-dependent protein kinase II in the gel was more than one order of magnitude higher than that in the gel containing no protein substrate.

Brain 14-3-3 protein is an activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+,calmodulin-dependent protein kinase II.

We have found that the 14‐3‐3 protein, an acidic neuronal protein, is substantially identical to the ‘activator’ protein [(1981) J. Biol. Chem. 256, 5404–5409] that activates tryptophan 5‐monooxygenase and tyrosine 3‐monooxygenase in the presence of Ca2+,calmodulin dependent protein kinase II. This finding is based on the remarkable similarity of both these proteins in physicochemical, biochemical and immunochemical properties, as well as on detection for the 14‐3‐3 protein of an activator activity towards tryptophan 5‐monooxygenase. The result suggests that the 14‐3‐3 protein plays a role in the regulation of serotonin and noradrenaline biosynthesis in brain.

Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain

Calcium ions, which are known to play important roles in the release and biosynthesis of neurotransmitters and other physiological functions of the nervous system [ 11, regulate the level of phosphorylation of a number of endogenous proteins in nerve terminals apparently through activation of a Ca’+sensitive protein kinase [2]. The involvement of a heat-stable Ca2+-sensitive regulator protein (calmodulin) in the Ca2+-sensitive protein phosphorylation in the nervous system has been suggested [3-6]. These studies have demonstrated that at least three distinct calmodulin-dependent protein kinases with different substrate specificities, one of which is involved in the regulation of tryptophan 5-monooxygenase, are present in rat brain cytosol.

Growth phase‐dependent changes in the subcellular localization of pre‐B‐cell colony‐enhancing factor1

A cDNA encoding the homolog of the human pre‐B‐cell colony‐enhancing factor (PBEF), a cytokine‐like secreted protein, was isolated from a rat cDNA library. This protein existed in both the cytoplasm and nucleus of the cells, and the amount was higher in the cytoplasm than in the nucleus of proliferating PC‐12 and Swiss 3T3 cells but higher in the nucleus than in the cytoplasm of the PC‐12 cells treated with nerve growth factor and the 3T3 cells grown to a confluent state. Thus, the so‐called PBEF is not a cytokine‐like secreted protein but an intracellular protein associated with the cell cycle.

Tyrosine 3-monooxygenase is phosphorylated by Ca2+-, calmodulin-dependent protein kinase, followed by activation by activator protein

Summary The activation of tyrosine 3-monooxygenase by Ca2+- calmodulin-dependent protein kinase in the presence of Ca2+, calmodulin, and ATP required the presence of activator protein. When tyrosine 3-monooxygenase was incubated with [γ-32P] ATP and Ca2+-, calmodulin-dependent protein kinase in the absence of the activator protein, the enzyme was phosphorylated but not activated. When the activator protein was added to the phosphorylated tyrosine 3-monooxygenase, the enzyme was activated. Thus, the activation of tyrosine 3-monooxygenase occurred in a two step reaction, phosphorylation by Ca2+-, calmodulin-dependent protein kinase and activation by activator protein.

Phosphorylation of microtubule-associated protein 2 by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues

Abstract Microtubule-associated protein 2 (MAP 2) from the rat brain was phosphorylated by calmodulin-dependent protein kinase (Kinase II) which occurs only in the brain tissues. The apparent Km for MAP 2 of Kinase II was 0.2 μ M . The maximum incorporation of phosphate into MAP 2 by the action of Kinase II was about 5 mol of phosphate per mol of MAP 2, while that by the action of cAMP-dependent protein kinase was about 3 mol of phosphate per mol of MAP 2. When microtubule-associated proteins were incubated with both Kinase II and cAMP-dependent protein kinase together, about 7 mol of phosphate were incorporated into 1 mol of MAP 2.

An immunohistochemical study of Ca2+/calmodulin-dependent protein kinase IV in the rat central nervous system : light and electron microscopic observations

We observed the distribution pattern of Ca2+/calmodulin-dependent protein kinase IV in rat brain and spinal cord using an immunohistochemical method by light and electron microscopy. Particularly strong immunoreactivity was detected in the telencephalic structures such as the olfactory bulb, cerebral cortex, hippocampal formation, caudate-putamen, most nuclei of the dorsal thalamus and the granule cell layer of the cerebellum. Relatively weak staining was observed in the amygdaloid body, some neuron groups of the brainstem reticular formation, the inferior olivary nucleus and the posterior horn of the spinal cord. Immunohistochemical reactivity was not detected in the globus pallidus, substantia nigra, sensory and motor nuclei of the cranial nerves, or in the spinal cord anterior horn. Overall, the distribution of Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity broadly paralleled the sites of expression of signals for messenger RNA of this enzyme. At the subcellular level, Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity appeared exclusively in the nuclei of neurons in the various brain regions, and immunopositive reactivity, although less strong, was also observed in dendritic processes, as well as on the granular endoplasmic reticulum in neuronal somata in these areas. Axon terminals, however, did not show immunoreactivity. These studies demonstrate that Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity is distributed widely in the central nervous system. The significance of the localization of this enzyme in nuclei is discussed in relation to gene expression.

A Novel Protein Phosphatase That Dephosphorylates and Regulates Ca2+/Calmodulin-dependent Protein Kinase II

A synthetic peptide corresponding to the autophosphorylation site of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (residues 281–289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn2+-dependent, and poly-l-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nm calyculin A or 10 μmokadaic acid. Mn2+, but not Mg2+, was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and α-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylasea (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca2+-independent activity of autophosphorylated CaMKII was reversed by the action of CaMKIIPase. Thus, CaMKIIPase appears to be a specialized protein phosphatase for the regulation of CaMKII.

Activation of tryptophan 5-monooxygenase by calcium-dependent regulator protein

Abstract Rat brainstem tryptophan 5-monooxygenase was activated about 2-fold by rat brain calcium-dependent regulator (CDR) protein. The activation required both ATP and Mg 2+ in the presence of low concentrations of Ca 2+ .

Identification of serotonergic neurons in human brain by a monoclonal antibody binding to all three aromatic amino acid hydroxylases

A monoclonal antibody, PH8, has been isolated and shown by immunocytochemistry to bind to serotonergic and catecholaminergic neurons in sections of the rat and human brain. In human brain, obtained at autopsy, particular fixation and embedding conditions eliminate the labelling of catecholaminergic neurons while leaving intact the labelling of serotonergic neurons. This property makes the antibody of potential use for structural studies of serotonergic neurons in the normal and diseased human brain. PH8 was raised to pure monkey phenylalanine hydroxylase and has been shown to bind to the 50,000 mol. wt. phenylalanine hydroxylase polypeptide. Immunocytochemical and immunochemical evidence is presented in support of the hypothesis that the labelling of serotonergic and catecholaminergic neurons results from the binding of PH8 to tryptophan and tyrosine hydroxylase, respectively.