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Characteristics of the F52 protein, a MARCKS homologue
P J Blackshear1, G M Verghese, J D Johnson
1Howard Hughes Medical Institute Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
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.
Insights
A newly identified mouse protein, F52, shows significant similarity to the Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein. This suggests F52 is a functional member of the MARCKS protein family, involved in cellular signaling pathways.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Protein Biochemistry
Background:
- The Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein is a key substrate for protein kinase C (PKC).
- Understanding MARCKS family proteins is crucial for deciphering cellular signaling pathways and their regulation.
Purpose of the Study:
- To characterize a newly cloned mouse cDNA, designated F52, and determine its relationship to the MARCKS protein family.
- To investigate the biochemical properties of the F52 protein, including its phosphorylation and calmodulin-binding capabilities.
Main Methods:
- Cloning and expression of mouse F52 cDNA in Escherichia coli.
- Biochemical assays to assess protein kinase C (PKC) phosphorylation and calmodulin binding.
- Peptide-based phosphorylation and binding studies.
- Two-dimensional electrophoresis for protein analysis.
Main Results:
- The F52 protein shares significant sequence homology with MARCKS, including key functional domains.
- Expressed F52 protein is a substrate for PKC and undergoes myristoylation.
- A synthetic peptide from F52's phosphorylation site effectively binds calmodulin and is phosphorylated by PKC with high affinity.
- F52 protein phosphorylation by PKC disrupts calmodulin binding.
Conclusions:
- The F52 protein is a novel member of the MARCKS protein family, exhibiting conserved biochemical properties.
- F52's functional attributes, including PKC phosphorylation and calmodulin interaction, suggest a role in cellular processes regulated by MARCKS proteins.