CONTROL OF CALCIUM AND PHOSPHORYLATION-REGULATED SIGNALLING PATHWAYS

钙调素（CaM）催化亚基的分子分析

Molecular analyses of the catalytic subunit of the calmodulin (CaM)- dependent protein phosphatase (PrP), calcineurin (CN), were extended with identification of a non-neural cDNA isoform in mouse testis encoded by a separate gene. Anti-peptide antibodies were prepared against the three mammalian genes and their alternatively-spliced isoforms; these recognize at least 10 charge variants on two-dimensional gel electrophoresis. Such complexity in the catalytic subunit of this phosphatase may reflect a need for substrate specific, Ca2+-dependent dephosphorylation. Homologues of CaM-PrP were cloned from fungi (Neurospora, Aspergillus) and from the cellular slime mold (Dictyostelium), indicating a role for Ca2+-regulated phosphoprotein metabolism in lower eukaryotes. Studies of their expression in these organisms suggested a relationship to morphogenic and developmental events. In Neurospora, mRNA and immunoreactive protein correlated with the extent of mycelial branching, whereas in Dictyostelium, increased mRNA accompanied differentiation into pre-stalk and pre-spore cells. In primary neuronal cultures examined by immunocytochemical methods, it also appeared that this enzyme may be localized to structures in developing neurites. A recombinant catalytic subunit from Neurospora was produced with a p-nitrophenol phosphatase activity comparable to that of brain enzyme, although it showed a much different phosphoprotein substrate specificity; chimeric constructs with "variable" regions taken from mammalian genes are being prepared to analyze these differences. Ultrastructural e.m. studies of phosphodiesterase (PDE) revealed high levels in post-synaptic regions of assymetric synapses, with little staining in other parts of the dendrite, consistent with a role for PDE in integration of afferent input. A 36 kDa neuron-specific CaM- associated protein (NCAP-36), having phosphorylated isoforms in vivo, was localized to the synaptoplasm of thalamic and midbrain structures and was not detected outside of brain. Peptide microsequencing of proteolytic digests of NCAP-36 showed no homologies to other classes of enzyme.