Autonomous activity and autophosphorylation of CAMPK-II in rat hippocampal slices: effects of tissue preparation.

Measurement of the proportion of calcium/calmodulin-stimulated protein kinase II (CaMPK-II) that is autonomously active or phosphorylated on Thr(286) is thought to provide an index of the degree to which CaMPK-II in a tissue has been activated. We have examined how various ways of handling hippocampal tissue can alter these properties. Both autonomous activity and phospho-Thr(286) content was high in freshly dissected hippocampus or freshly cut hippocampal slices. After incubation of hippocampal slices in artificial cerebrospinal fluid for 120 min, both properties of CaMPK-II decreased to a steady state level. Freeze-thaw or cutting the equilibrated slices could rapidly increase both autonomous activity and phospho-Thr(286) immunoreactivity of CaMPK-II. These increases were comparable to changes induced by experimental treatment. Therefore, our results suggest that considerable care needs to be taken over the way in which hippocampal slices are handled.

In vitro phosphorylation of medial vestibular nucleus and prepositus hypoglossi proteins during behavioural recovery from unilateral vestibular deafferentation in the guinea pig.

Unilateral removal of vestibular nerve input to the vestibular nuclei (e.g. by unilateral labyrinthectomy, UL) results in severe ocular motor and postural disorders which disappear over time (vestibular compensation). We investigated whether recovery of ocular motor function is temporally correlated with changes in protein phosphorylation in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PH; MVN/PH) in vitro. Bilateral MVN/PH were dissected from 48 guinea pigs following decapitation at 10 h, 53 h or 2 weeks post-UL, or -sham operation and frozen. Tissue extracts were incubated with [gamma-32P]ATP +/- Ca2+ plus phorbol 12,13-dibutyrate and phosphatidylserine. UL resulted in a significant bilateral increase in the 32P-incorporation into a 65-85 kDa band (probably the myristoylated alanine-rich C kinase substrate, MARCKS) in compensated animals (53 h post-UL) under conditions which favoured the activation of protein kinase C. Under identical conditions, the labelling of a 42-49 kDa protein (P46) was increased significantly in the bilateral MVN/PH between either 10 h or 53 h and 2 weeks post-UL; there were no significant changes over time in sham controls. These results show that later stages of vestibular compensation are accompanied by changes in the phosphorylation of several likely protein kinase C substrates in the MVN/PH in vitro.

Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.

Developmental changes in phosphorylation of MAP-2 and synapsin I in cytosol and taxol polymerised microtubules from chicken brain.

In cytosol, cyclic AMP stimulated phosphorylation of microtubule associated protein-2 (MAP-2) increased from 2 days to adult in proportion to the increase in the concentration of MAP-2. By contrast, the calmodulin stimulated phosphorylation of MAP-2 decreased in proportion to the decrease in the concentration of calmodulin stimulated protein kinase II (CMK II). Similarly, the cAMP stimulated phosphorylation of the site on synapsin I labeled by the cAMP stimulated protein kinase (PKA) changed little during development whereas the calcium/calmodulin stimulated phosphorylation of the CMK II site decreased dramatically in proportion to the decrease in the concentration of CMK II. The decrease in the concentration of CMK II which occurs in cytosol during synapse maturation was also observed in taxol polymerised microtubules and the effects of the change in the relative concentrations of CMK II and PKA on the phosphorylation of MAP-2 and synapsin I in this fraction were similar to that observed in the cytosol. These results are consistent with the hypothesis that the developmental changes in phosphorylation of endogenous substrates by PKA is controlled largely by changes in the concentration of those substrates, whereas the concentration of CMK II is limiting so that the developmental changes in the phosphorylation of endogenous substrates by CMK II are a function of the concentration of CMK II itself as well as the concentration of endogenous substrates. Some possible functional consequences of this during synapse maturation are discussed.

Purification and characterization of calmodulin-stimulated protein kinase II from two-day and adult chicken forebrain.

Soluble calmodulin-stimulated protein kinase II has been purified from 2-day and adult chicken forebrain. At both ages the holoenzyme eluted from a Superose-6B column with an apparent molecular weight of approximately 700,000 daltons and contained three subunits. The subunits were found to be the counterparts of the alpha, beta, and beta' subunits of the enzyme purified from adult rat brain in that they had one-dimensional phosphopeptide maps that were indistinguishable from those of the corresponding subunit in the rat enzyme and they migrated in SDS-polyacrylamide gels with the same apparent molecular weights. However, the doublet formed by the beta subunit was much more clearly resolved in the chicken enzyme and the beta' subunit, which was much more abundant in the adult chicken than in the adult rat, was also found to be a doublet. The ratio of the concentrations of the alpha and beta subunits changed during development. By autoradiography following autophosphorylation, the alpha:beta ratios of the 2-day and adult enzymes were 0.89 +/- 0.07 and 1.92 +/- 0.26, respectively; by silver staining the alpha:beta ratios were 0.95 +/- 0.11 and 1.85 +/- 0.17, respectively. The concentration of the beta' subunit was equal to that of the beta subunit at both ages. Autophosphorylation produced a decrease in the electrophoretic mobility of the alpha and beta subunits in SDS-polyacrylamide gels and a marked decrease in the calcium dependence of the substrate phosphorylation activity of the enzyme at both ages. The purified enzyme from chicken brain appeared to be more stable under standard in vitro assay conditions than the rat enzyme, and this was particularly so for the enzyme from 2-day forebrain.

The subcellular distribution of a membrane-bound calmodulin-stimulated protein kinase.

Incubation of subcellular fractions isolated from rat cerebral cortex with [gamma-32P]ATP results in the phosphorylation of a number of proteins including two with apparent molecular weights of approximately 50,000 and 60,000 daltons. These phosphoproteins were shown to be the autophosphorylated subunits of a calmodulin-stimulated protein kinase by a number of physicochemical criteria, including their mobility on non-equilibrium pH gradient electrophoresis, their phosphopeptide profiles and phosphorylation characteristics. When a crude membrane fraction obtained following osmotic lysis of a P2 fraction was labeled and subsequently fractionated on sucrose density gradients, approximately 80% of the autophosphorylated kinase was associated with fractions enriched in synaptic plasma membranes. Other substrates of calmodulin kinase(s) were similarly distributed. Detergent extraction of synaptic plasma membranes to produce synaptic junctions and post-synaptic densities indicated that the majority of the autophosphorylated kinase was solubilized, apparently as a holoenzyme. The major post synaptic density protein (mPSDp) was not readily extracted by detergents and was largely unlabeled under the conditions used for phosphorylation, and yet this protein is structurally closely related to the kinase subunit. It is possible that this lack of labeling is due to the mPSDp being attached to the PSD in a different way or being present there in a different isoenzymic form from that of the readily autophosphorylated enzyme subunit. Thus, the data suggest that, in vitro at least, a number of pools of calmodulin kinase exist in neuronal membranes.

The maturation of post-synaptic densities in chicken forebrain.

During normal post-hatch development and maturation in chicken forebrain there was a doubling in the average thickness of the post-synaptic densities (PSDs) in isolated synaptosomes, but no change in the average length of synaptic junctions. In the same period the concentration of the major post-synaptic density protein ( mPSDp ) in membranes prepared from the synaptosomes increased 3-5-fold. We suggest that immature synapses have a basic PSD framework which contains little or no mPSDp and that, during development, specific components such as the mPSDp are progressively incorporated into it. We suggest that the thickness of the PSD is proportional to the amount of mPSDp it contains.

The major calmodulin-stimulated phosphoprotein of synaptic junctions and the major post-synaptic density protein are distinct.

The major post-synaptic density protein (mPSDp) present in isolated synaptic junction fractions is distinct from the major phosphoprotein (50Kpp) that is labelled by an endogenous calmodulin-stimulated protein kinase. mPSDp and the 50Kpp have different apparent molecular weights on sodium dodecyl sulphate polyacrylamide gels and the presence of 50Kpp in brain soluble fractions indicates that the two proteins have different subcellular distributions.