Characteristics of the F52 protein, a MARCKS homologue.
A recently cloned mouse cDNA designated F52 encodes a putative protein with striking sequence similarity to the MARCKS protein, a major cellular substrate for protein kinase C (PKC). Major regions of sequence similarity include the amino-terminal myristoylation consensus sequence and the central calmodulin-binding/PKC phosphorylation site domain. The F52 protein was expressed in Escherichia coli with apparent M(r) 50,000; it was a substrate for PKC and comigrated on two-dimensional electrophoresis with a myristoylated protein whose phosphorylation was stimulated by phorbol 12-myristate 13-acetate in mouse neuroblastoma cells. The F52 protein also was myristoylated in E. coli by co-expression with N-myristoyltransferase. A 24-amino acid peptide derived from the protein's phosphorylation site domain was a good substrate for PKC; like the cognate MARCKS peptide, it was phosphorylated with high affinity (S0.5 = 173 nM) and positive cooperativity (KH = 5.4). The F52 peptide also bound calmodulin with high affinity (Kd = less than 3 nM); this binding could be disrupted by phosphorylation of the peptide with PKC, with a half-time of 8 min. The F52 protein is clearly a member of the MARCKS family as defined by primary sequence; in addition, the two proteins share several key attributes that may be functionally important.
Blackshear
P J
PJ
Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Verghese
G M
GM
Johnson
J D
JD
Haupt
D M
DM
Stumpo
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051379:Mice; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013755:Tetradecanoylphorbol Acetate / Q000737:chemistry; D014407:Tumor Cells, Cultured
D002148:Calmodulin-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; C512945:Marcksl1 protein, mouse; D008565:Membrane Proteins; D008840:Microfilament Proteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D013755:Tetradecanoylphorbol Acetate