Mouse MARCKS is a prominent myristoylated alanine-rich C kinase substrate implicated in brain development, calcium/calmodulin signaling, and membrane cytoskeletal restructuring, and is developmentally regulated in a cell- and tissue-specific fashion. In this study, transcriptional regulation of mouse MARCKS promoter in the neuronally derived immortalized hippocampal cells (HN33) was examined for a portion of 5'-flanking genomic sequence from -993 to + 1 relative to the translation start site. Transfection experiments carried out in this neural cell line identified, for the first time, that the distal promoter segment from -993 to -713 plays a crucial role as an enhancer/activator element in the up-regulation of the basal transcription activity driven by MARCKS core promoter sequence. Motif analyses revealed at least 12 overlapping potential transcription factor binding sites in this region, among which a prominent GA-rich sequence centered at -765 has been shown to be functionally important in the binding of Sp1 protein-like complex. Deletion of the GA-rich segment significantly reduced the MARCKS promoter activity. Further, competitive EMSA indicated two additional sites within the -993/-713 that may also interact with Sp1 protein, demonstrating that the activator function of -993/-713 is under control of multiple Sp1 transcription factors. Unlike the distal promoter sequence, the proximal core promoter sequence (-649/-438) contains a GC-rich box and a Z-DNA-forming segment and is critical to basal transcription. The deletion of -649/-438 segment has been shown to drastically impair the promoter activity even in the presence of -993/-713, suggesting that its presence is also important to the function of -993/-713. These data emphasize that the synergistic interaction between distal and proximal promoter sequences is indispensable for the optimal MARCKS promoter function in the immortalized hippocampal cells. The discovery of the activator function of the MARCKS distal promoter region, and its potential interaction with multiple Sp proteins may provide a new clue to the understanding of Macs transcriptional regulation in brain. (C) 2002 Elsevier Science (USA).