lonomycin-regulated phosphorylation of the myeloid calcium-binding protein p14

Abstract

Two associated calcium-binding proteins (CaBPs) have recently been identified specifically in cells of myeloid origin. These proteins have relative molecular masses (M_r) of 8,000 and 14,000 and are variously referred to as the cystic fibrosis antigen¹, the LI light chain², MRP-8 (ref. 3) or p8, and the LI heavy chain², MRP14 (ref. 3) or pi4, respectively. The expression of p8 and p14 seems to be confined to a specific stage of myeloid cell differentiation, because both proteins are expressed in circulating neutrophils and monocytes but not in normal tissue macrophages^4,5. In chronic inflammatory conditions, however, such as rheumatoid arthritis, macrophages in affected tissues express both p8 and p14 (refs 4, 6, 7). These proteins are members of a family of CaBPs of low M_r (ref. 8), which include S-100 α and β proteins^9,10, calcyclin (2A9)¹¹, intestinal CaBP¹² and p11 (ref. 13). All the proteins have an M_r of ∼10,000 with the exception of p14 which has a longer C-terminal sequence after the second calcium-binding domain. Little is known about their function, although by analogy with calmodulin they could be molecules involved in intracellular signalling that are activated by an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) (ref. 14). Here we report that p14 is phos-phorylated in both monocytes and neutrophils. The level of p14 phosphorylation can be increased by elevating the [Ca²⁺]₁ using the ionophore ionomycin, but is not affected by activation of protein kinase C using phorbol 12,13-dibutyrate. The phosphorylated residue is threonine at position 113, which is the penultimate amino acid in p14 and contained in the longer 'tail' sequence. Part of this sequence is identical¹⁵ to the neutrophil immobilizing factors NIF-1 and NIF-2 (ref. 16), indicating that the phosphorylation event could have a role in the generation of NIF activity in the p14 protein.