COMPLETE PRIMARY STRUCTURE AND PHOSPHORYLATION SITE OF THE 65-KDA MACROPHAGE PROTEIN PHOSPHORYLATED BY STIMULATION WITH BACTERIAL LIPOPOLYSACCHARIDE

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-kD a cytosolic protein (pp65) that was phosphorylated specifically in LPS -stimulated murine macrophages. In the present study, the complete pri mary structure of pp65 was determined on the basis of the cDNA contain ing 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 cel ls, and that it contains a unique series of Ca2+, calmodulin, and acti n binding domains. A single phosphorylated peptide was isolated from t he tryptic digest of pp65 by reverse-phase HPLC. From the amino acid s equence of the dodecapeptide Gly-Ser-Val-Ser-Asp-Glu-Glu-Met-Met-Glu-L eu-Arg, the phosphorylation site of pp65 was located at the N-terminal region adjacent to the first Ca2+ binding domain. This sequence conta ins a repeat of the casein kinase II motif Ser-Xxx-Xxx-Glu/Asp and, to gether with the preceeding Arg residue, constitutes the consensus sequ ence Arg-Xxx-Ser for cAMP-dependent protein kinase (PKA) and protein k inase C (PKC), but no mitogen-activated protein kinase (MAPK)-specific motif is found. These results, taken together with previous observati ons on the process of macrophage activation by LPS, demonstrate that p p65 is phosphorylated by an LPS-induced protein kinase other than MAPK and exerts its function on the cytoskeleton in a Ca2+/calmodulin-depe ndent manner.