The protein kinase C (PKC) substrate GAP-43 is already expressed in neural precursor cells, colocalizes with PKCη and binds calmodulin

Abstract

Expression of the growth-associated protein of 43-kDa (GAP-43), which is described as a postmitotic, neuron-specific major protein kinase C (PKC) substrate, was investigated in the murine embryonic carcinoma cell line PCC7-Mz1 which develops into a brain-tissue-like pattern of neuronal, fibroblast-like and astroglial cells upon stimulation with all-trans retinoic acid (RA). GAP-43 expression was very low in stem cells, but increased on mRNA and protein level within the 12 h after differentiation was initiated. While the P1 promoter of the GAP-43 gene gave rise to a 1.6-kb mRNA and was already active at a very low level in PCC7-Mz1 stem cells, transcription of the P2 promoter, which resulted in a 1.4-kb mRNA, was completely blocked in stem cells but increased rapidly after RA treatment. Within the first 2 days of neural differentiation, GAP-43 was localized with the cytoplasmic membrane and the Golgi complex of proliferating neural precursor cells. Then, GAP-43 was translocated to the growth cones and neurites, and from day 6, when neurons began to acquire polarity, the protein was found in the axons. GAP-43 was never detected in the non-neuronal PCC7-Mz1 derivatives, i.e. in fibroblasts or glial cells. In the foetal rat brain (prenatal day F11), GAP-43 was expressed in the optic stalk, the lense plakode and in the postmitotic neurons of the marginal zone of the hindbrain. Moreover, in a layer between the ventricular and marginal zone of the hindbrain (F13) and forebrain (F15), GAP-43 was already expressed in mitotic neural precursor cells. In PCC7-Mz1 cultures, 2 days after addition of RA, GAP-43 became phosphorylated upon activation of PKC, and colocalized specifically with the novel PKC isoform η. Phosphorylation of GAP-43 caused a disruption of its complex with calmodulin. These data demonstrate that GAP-43 is already a functional PKC substrate in prolific neuronal precursor cells, and may participate in neuronal cell lineage determination.