CALCIUM-DEPENDENT PHOSPHORYLATION OF GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) IN THE RAT HIPPOCAMPUS - A COMPARISON OF THE KINASE PHOSPHATASEBALANCE IN IMMATURE AND MATURE SLICES USING TRYPTIC PHOSPHOPEPTIDE MAPPING/

In previous work we showed that phosphorylation of the astrocyte marke r glial fibrillary acidic protein (GFAP) in hippocampal slices from ad ult rats is dependent on external Ca2+, whereas in slices from immatur e rats aged 12-16 days postnatal P-32 incorporation into GFAP is inhib ited by external Ca2+. The nature of this late developmental change in Ca2+ sensitivity for GFAP phosphorylation was investigated in the pre sent work by comparing in immature and adult animals phosphorylation o f GFAP by endogenous protein kinase activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with [gamma-P-32]ATP and from slices labelled with [P-32]phos phate. Cytoskeletal fractions prepared from immature and adult hippoca mpus both contained endogenous protein kinase activity towards GFAP an d other proteins stimulated by Ca2+/calmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca2+/calmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from t hese fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phospho rylated by Ca2+/calmodulin-stimulated kinase activity. Maps derived fr om GFAP isolated from adult slices labelled in the presence of Ca2+ an d immature slices labelled in the absence of Ca2+ were qualitatively i dentical, with minor differences from the cytoskeletal maps. At both a ges the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca2+/calmodulin kinase in the cytoskeletal fraction s. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylate d in vitro by CaM kinase II, suggesting that even in the absence of ex ternal Ca2+, kinase activity directly or indirectly dependent on Ca2was occurring in the immature slices. The near identity of the phospho rylation sites at the two ages suggest that the change in Ca2+ sensiti vity of GFAP phosphorylation during development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism(s) whereby Ca2+ controls the relative activity of these enzymes. (C) 1997 Elsevier Science B.V.