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Complete primary structure and phosphorylation site of the 65-kDa macrophage protein phosphorylated by stimulation
H Shinomiya1, A Hagi, M Fukuzumi
1Department of Microbiology, Ehime University School of Medicine, Japan.
Abstract:
There is ample evidence that intracellular protein phosphorylation is a mandatory event in the process of macrophage activation by LPS, yet how this event is initiated and what roles the phosphorylated proteins are assigned to are poorly understood. We previously isolated a 65-kDa cytosolic protein (pp65) that was phosphorylated specifically in LPS-stimulated murine macrophages. In the present study, the complete primary structure of pp65 was determined on the basis of the cDNA containing an open reading frame of 1881 bases. The sequence of pp65 revealed that it is a murine homologue of human L-plastin, recently identified as a novel transformation-induced polypeptide of neoplastic human cells, and that it contains a unique series of Ca2+, calmodulin, and actin binding domains. A single phosphorylated peptide was isolated from the tryptic digest of pp65 by reverse-phase HPLC. From the amino acid sequence of the dodecapeptide Gly-Ser-Val-Ser-Asp-Glu-Glu-Met-Met-Glu-Leu-Arg, the phosphorylation site of pp65 was located at the N-terminal region adjacent to the first Ca2+ binding domain. This sequence contains a repeat of the casein kinase II motif Ser-Xxx-Xxx-Glu/Asp and, together with the preceeding Arg residue, constitutes the consensus sequence Arg-Xxx-Ser for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), but not mitogen-activated protein kinase (MAPK)-specific motif is found. These results, taken together with previous observations on the process of macrophage activation by LPS, demonstrate that pp65 is phosphorylated by an LPS-induced protein kinase other than MAPK and exerts its function on the cytoskeleton in a Ca2+/calmodulin-dependent manner.
Insights
Researchers identified a protein (pp65) crucial for macrophage activation by LPS. This protein, a murine L-plastin homolog, is phosphorylated by specific kinases and interacts with the cytoskeleton in a calcium-dependent manner.
Area of Science:
- Immunology and Cell Biology
- Molecular Biology
Background:
- Macrophage activation by lipopolysaccharide (LPS) involves intracellular protein phosphorylation, but the initiating events and roles of these proteins are unclear.
- A 65-kDa cytosolic protein (pp65) was previously identified as being phosphorylated specifically in LPS-stimulated murine macrophages.
Purpose of the Study:
- To determine the complete primary structure of pp65 and identify its phosphorylation site and regulatory mechanisms.
- To elucidate the functional role of pp65 in LPS-induced macrophage activation.
Main Methods:
- Determined the complete primary structure of pp65 using cDNA sequencing.
- Isolated and sequenced a phosphorylated peptide from pp65 tryptic digest using reverse-phase HPLC.
- Analyzed the phosphorylation site for kinase consensus motifs.
Main Results:
- pp65 is the murine homolog of human L-plastin, possessing Ca2+, calmodulin, and actin binding domains.
- The phosphorylation site was localized to the N-terminal region, adjacent to a Ca2+ binding domain, containing casein kinase II and PKA/PKC consensus motifs.
- pp65 is phosphorylated by an LPS-induced protein kinase distinct from MAPK.
Conclusions:
- pp65 plays a role in the cytoskeleton during LPS-induced macrophage activation.
- Its function is regulated by Ca2+/calmodulin-dependent mechanisms and phosphorylation by specific kinases.