Emeritus Professor John Rostas

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. © 1989 Humana Press Inc.

The net level of cyclic AMP-stimulated protein phosphorylation was investigated in cytosolic and membrane fractions from chicken forebrain between embryonic day 13 (E13) and 52 days post-hatching. Throughout this period the majority of the net level of cAMP-stimulated phosphorylation of endogenous proteins was in the cytosolic fractions. Between day -8 (E13) and adult, the net level of cAMP-stimulated phosphorylation of endogenous proteins in the cytosol (S3) and crude synaptic plasma membrane (P2-M) fractions fell by 3 and 4 fold, respectively, when expressed per mg protein and rose by 5 and 10 fold, respectively, when expressed per fraction. The changes in specific activity were completed by 6-15 days post-hatching. The occluded cytosol (P2-S) fraction showed little change in the net level of cAMP-stimulated phosphorylation of endogenous proteins per mg protein. Major changes in phosphoprotein patterns involving both decreases and increases in phosphorylation occurred in all fractions from day -8 (E13) to day 6 post-hatch; thereafter the phosphoprotein bands and their relative intensities were unchanged. Three bands (P90 in S3; P41 and P31 in P2-M) contained major cAMP-stimulated phosphoproteins in embryonic brain but were absent after hatching. When cAMP-stimulated phosphorylation activity was measured in S3 and P-2M using an exogenous peptide substrate (Kemptide) there was no change in kinase activity per mg protein between day -8 (E13) and 30 days post-hatch. This suggests that the decrease in the net level of cAMP stimulated phosphorylation of endogenous proteins was due to the decrease in levels of endogenous phosphoproteins rather than protein kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The development of calmodulin stimulated protein phosphorylation, with particular reference to calmodulin-stimulated protein kinase II (CMK II), was investigated in 3 subcellular fractions of chicken forebrain: cytosol (S3), crude synaptic plasma membranes (P2-M) and occluded cytosol (P2-S). Changes in the level of calmodulin-stimulated phosphorylation of endogenous proteins occurred over a protracted time course and were not complete until after day 52 post-hatching. By day 15 post-hatching, calmodulin-stimulated phosphoproteins characteristic of embryonic fractions had all disappeared and those characteristic of adult tissue were present but not necessarily at their mature levels. The levels of CMK II were estimated from the autophosphorylation of the alpha-subunit which was the only phosphoprotein present at 53,000 Da in the 3 fractions. Overall, calmodulin-stimulated phosphorylation and CMK II levels were low in embryonic brain and high in adult brain but two specific changes in CMK II were observed during development: (1) although CMK II concentrations increased in both membrane and cytosolic fractions until day 23 the kinase was predominantly cytoplasmic (approximately 75%) until day 23, after which it became increasingly membrane bound so that by day 52 post-hatching the majority of CMK II was present in the synaptic membrane fraction, and (2) the relative concentrations of the alpha- and beta-subunits changed from an alpha:beta-value of approximately 1:1 in the 19 day embryo to approximately 1:2 by 15 days post-hatch after which no further change was seen. The occurrence of major changes in the calmodulin stimulated protein phosphorylation system for up to 6-8 weeks after synapse formation is completed in the forebrain, provides further support for the existence of a synapse maturation phase of neuronal differentiation which is distinct from synapse formation. This phase involves only a specific subset of the developmental changes occurring in the calmodulin-stimulated phosphorylation system.

We have used the method of Austin, Lowry, Brown and Carter, to measure the steady-state metabolic half-life of tubulin (alpha and beta individually) and actin (beta and gamma together) in the total cytosolic (S3), microsomal (P3), synaptic plasma membrane (SPM) and synaptic junction (SJ) subcellular fractions from 6-day-old and adult chicken forebrain. In the SPM and SJ fractions we also measured the steady-state metabolic half-life of the major postsynaptic density protein (mPSDp). In SPM and SJ fractions from 6-day-old chickens tubulin and actin turned over approximately twice as slowly (t1/2 approximately equal to 24 days) as tubulin and actin in the S3 fraction (t1/2 approximately equal to 13 days). This difference was unlikely merely to be due to association with membranes since the t1/2 values for the proteins were the same in P3 and S3. The estimated t1/2 values for mPSDp were similar to that for tubulin and actin in SPM and SJ fractions. Similar results were obtained in adult chickens except that all t1/2 values in all fractions were approximately 30% larger. The calculated t1/2 values did not change between labelling periods of 4 and 6.5 h suggesting that the lag phase of incorporation of newly synthesized PSD proteins is sufficiently rapid to not produce this result artefactually. When the brain from a non-labelled chicken was homogenized in the presence of the S3 fraction from a labelled chicken and sub-fractionated the relative specific activities of the SPM and SJ fractions produced were 1-2% of those from the labelled brain. These results support the notion that tubulin and actin are intrinsic components of the PSD.

The postnatal development of calmodulin-stimulated phosphorylation of endogenous proteins, in particular the autophosphorylated subunits of the calmodulin-stimulated protein kinase II, were investigated in subcellular fractions of rat cerebral cortex. The major subunit had a mol. wt. of 53,000 Da (designated 50 kDa) and the minor one a mol. wt. of 63,000 Da (designated 60 kDa). The 50-kDa subunit was found to be the only significant phosphoprotein in each fraction and throughout development at its molecular weight. However, the 60-kDa subunit was found to comigrate with other phosphoproteins that accounted for up to 15% of the radioactivity at this molecular weight and which differed between the fractions. 50-kDa autophosphorylation was found to be 3-fold greater in cytoplasmic fractions at day 10 and by adults was evenly distributed between membrane and cytoplasmic fractions. A similar pattern was also found for the total calmodulin-stimulated phosphorylation. Changes in autophosphorylation activity of the 50-kDa subunit were found to represent changes in kinase activity rather than alterations in phosphatase activity. In the membrane, this change was shown to be due to changes in the amount of enzyme. Although in the adult autophosphorylation activity is evenly distributed between membrane and soluble fractions, when differences in phosphatase activity and lack of autophosphorylation activity of the majority of post-synaptic density-associated kinase is taken into account, it is clear that the vast majority of the enzyme is membrane-bound. Phosphorylation of endogenous substrates paralleled the development of 50-kDa subunit autophosphorylation, most of which occurred between day 14 and day 30, a period which follows the most rapid phase of synaptogenesis. This pattern was different from that of the phosphorylation of myelin basic protein and two substrates of the calcium-phospholipid-dependent protein kinase. There was also a change in the ratio of autophosphorylation activity of the 50-kDa and 60-kDa subunits during development which appears to be due to a change in the amount of the subunits themselves. This ratio was the same in all fractions at any one age. We suggest that this change is due to the existence of at least two developmentally regulated isoenzymes in the cortex.