Abstract
The expression of a major cellular substrate for protein kinase C, the MARCKS protein, is regulated in a cell-, tissue-, and developmental stage-specific fashion; in addition, this expression can be stimulated acutely by various cytokines in certain cell types. We have begun to characterize the human gene in order to elucidate the genetic elements responsible for this highly regulated expression. We first cloned a human MARCKS cDNA, which encoded a predicted protein of 332 amino acids (Mr 31,600) that was approximately 89, 74, and 59% identical to the bovine, mouse, and chicken proteins, respectively. Regions conserved at the amino acid level included the amino-terminal myristoylation consensus sequence, the site of intron splicing, and the phosphorylation site domain. The human cDNA was used to demonstrate that tumor necrosis factor-alpha could rapidly stimulate MARCKS gene transcription in the human promyelocytic leukemia cell line HL60. Genomic clones were then isolated; sequence analysis identified a putative promoter region that had no TATA box and contained multiple transcription initiation sites in a region spanning 57 base pairs (bp). This was followed by a 5'-untranslated region of approximately 400 bp, which displayed a complex predicted secondary structure with a delta G of -73.4 kcal/mol. Plasmid constructions containing between 52 and 1453 bp of the human MARCKS promoter linked to the human growth hormone gene were then used in transient expression experiments. Constructions containing 52 and 110 bp of the MARCKS promoter did not exhibit promoter function while the larger constructions all exhibited promotor function; the 248-bp fragment of the MARCKS promoter was 80% as effective as the human ferritin promoter in stimulating expression of human growth hormone in intact cells. Using an insert from the human genomic clone as a probe, we identified human chromosome 6, q21-qter, as the location of the MARCKS gene; this has been assigned the gene symbol MACS.
Highlights
14263, and the **Cancer Research Campaign, Department of Biochemical Genetics, Paterson Institutefor Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, United Kingdom M20 9BX
We have begun to characterize the human gene in order to elu-Protein kinase C (PKC)’ has beenfound to play an imporcidate the genetic elements responsible for this highlytant role in a wide array of cellular processes [1].Since its regulateedxpression
Regions conserved at mediating PKC’s cellular effects. One such cellular substrate the amino acid level included the amino-terminal myrf-or PKC, the myristoylated alanine-rich C kinase substrate, istoylation consensus sequencet,he siteof intron splic- or MARCKS protein (a),has been studied in our laboratory ing, and the phosphorylationsite domain
Summary
Of this elementwill be necessary to test thispossibility. Ourstudiesalso suggest that TNF-a can stimulate the. The MACS promoter is interesting in that it appears to stimulated MACS transcription by transfectionstudiesin lack a TATA box, typical of many housekeeping gene pro- macrophage cell lines That this induction is likely to be of moters [28], and yet apparently diretcistsue-specific expres- physiological importance was suggested by recent studies in sion of the gene [2, 3, 23]. The first (class 1)includes GC- We initially hoped that the LM-TK- cell line used in our rich promoters, characterizedby their: 1)appearance primar- transfectionstudies would be usefulfor studies of MACS ily in housekeeping genes; 2) multiple transcription initiation tissue-specific expression because, unlike other fibroblastcell sites; and 3) several potential binding sites for transcription lines tested, thesecells lackdetectable MARCKS mRNA and factor S p l [31].The second (class 2) is characterized by the protein [2].
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