D. Tinelli

cAMP-dependent phosphorylation of soluble and crude microtubule fractions of rat cerebral cortex after prolonged desmethylimipramine treatment

We have analyzed the cAMP-dependent phosphorylation system in the cerebral cortex and hippocampus of rats after acute and chronic administration of desmethylimipramine. Prolonged desmethylimipramine administration modified the cAMP-dependent endogenous phosphorylation of a protein band with apparent molecular weight 280 kDa from the cerebrocortical-soluble fraction. The effect appeared to be specific and associated with the chronic but not the acute administration of desmethylimipramine since we did not obtain any modification in other endogenous cAMP phosphoproteins of either the particulate or soluble fraction of the cerebral cortex. 280 kDa was identified as the soluble microtubule associated protein 2 on the basis of molecular weight, endogenous phosphorylation and immunological recognition. Prolonged desmethylimipramine administration did not induce any modification in the soluble cAMP-dependent endogenous phosphorylation of 280 kDa in other brain areas such as hippocampus, striatum or cerebellum, suggesting a region-specific effect of chronic desmethylimipramine treatment. Microtubule-associated protein 2 is a neuronal protein highly enriched in the dendritic portion of neurons and represents one of the major substrates in the cell for the type II cAMP protein kinase. Since the type II cAMP protein kinase that catalyzes the phosphorylation of microtubule-associated protein 2 copurifies with microtubules, we performed endogenous phosphorylation using increasing concentrations of cAMP in a crude microtubule preparation where microtubule-associated protein 2 appeared to be more concentrated. Under our conditions the maximal effect occurred at 1 microM cAMP, revealing increased 32P incorporation in microtubule-associated protein 2 from a crude microtubule preparation obtained from the cerebral cortex of rats treated with desmethylimipramine. Photoaffinity labelling with 8-azido-[32P]cAMP of the various fractions obtained during the preparation of crude microtubules (S1, S2 and crude microtubules) revealed an increase in the labelling of a protein band with apparent molecular weight of 52 kDa after desmethylimipramine treatment. The labelling of a 47 kDa protein band, which is also present in S1 and S2 fractions was, however, not altered by drug treatment. In conclusion, our studies demonstrated that prolonged desmethylimipramine treatment elicited specific changes in the phosphorylation system associated with a crude microtubule fraction.

Evidence for the existence of cAMP-dependent protein kinase phosphorylation system associated with specific phosphoproteins in stable microtubules from rat cerebral cortex

Cyclic AMP is a second messenger by which different extracellular signals are transduced into biological responses. Within the cell, most of the effects of cAMP are mediated through the cAMP protein kinase which appears to be localized in specific compartments of the cell near to their substrate proteins. In the present study, we have investigated the possible association of cAMP-dependent protein kinase, its substrate proteins and RII binding proteins in stable microtubules from rat cerebral cortex. The results show that in this fraction there is a cAMP binding protein of 52-54 kDa. This cAMP receptor is in the inactive holoenzyme form, since the addition of cAMP (5 microM) induces an increase in the endogenous phosphorylation of different stable microtubules polypeptides, which is completely inhibited in the presence of a specific protein kinase inhibitor (PKI 5-24 1 microM). Interestingly, overlay binding assay reveals that beside MAP2, 32P/R II is able to bind stable microtubule proteins of M(r) 150 and 75 kDa which, according to their electrophoretic mobility, can also be endogenous substrates for the enzyme. We conclude that cAMP-dependent phosphorylation system is indeed associated with stable microtubules from rat cerebral cortex.