The protein kinase C (PKC) substrate GAP-43 is already expressed in neural precursor cells, colocalizes with PKCeta and binds calmodulin.
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 eta. 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.
Laboratory of Molecular Neurobiology, Mainz, Germany.
C
C
Esdar
S A
SA
Oehrlein
S
S
Reinhardt
A
A
Maelicke
T
T
Herget
D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology; D002147:Calmodulin / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D015342:DNA Probes; D005455:Fluorescent Antibody Technique; D019922:GAP-43 Protein / Q000032:analysis / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D006056:Golgi Apparatus / Q000201:enzymology; D019394:Ki-67 Antigen / Q000032:analysis / Q000235:genetics; D051379:Mice; D014411:Neoplastic Stem Cells; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013234:Stem Cells / Q000166:cytology / Q000201:enzymology*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000201:enzymology
D002147:Calmodulin; D015342:DNA Probes; D019922:GAP-43 Protein; D019394:Ki-67 Antigen; D012333:RNA, Messenger; D011493:Protein Kinase C