Macrophage colony-stimulating factor (M-CSF or CSF-1) controls the development of macrophage lineage cells via activation of its tyrosine kinase receptor, c-Fms. After adding CSF-1 to M1 myeloid cells expressing CSF-1R (CSF-1 receptor), tyrosine phosphorylation of many cellular proteins occurs, which might be linked to subsequent macrophage differentiation. The biological significance and characterization of such proteins were explored by a dual strategy comprising two-dimensional SDS/PAGE analysis of cell lysates of CSF-1-treated M1 cells expressing the wild-type or a mutated receptor, together with an enrichment strategy involving a tyrosine-phosphorylated receptor construct. In the present study, we report the identification by MS of a novel, low-abundance, 110 kDa form of myosin XVIIIA (MysPDZ, myosin containing PDZ domain), which appears to be preferentially tyrosine-phosphorylated after CSF-1R activation when compared with other known isoforms. Receptor mutation studies indicate that CSF-1R-dependent tyrosine phosphorylation of p110myosin XVIIIA requires Tyr-559 in the cytoplasmic domain of the receptor and is therefore Src-family kinase-dependent. Gelsolin, Erp61 protein disulphide-isomerase and possibly non-muscle myosin IIA were also tyrosine-phosphorylated under similar conditions. Similar to the more abundant p190 isoform, p110 myosin XVIIIA lacks a PDZ domain and, in addition, it may lack motor activity. The phosphorylation of p110 myosin XVIIIA by CSF-1 may alter its cellular localization or target its association with other proteins.
Abbreviations used: BMM, bone-marrow-derived macrophage; CLB, cell lysis buffer; CSF, colony-stimulating factor; CSF-1R, CSF-1 receptor; M-CSF, macrophage CSF; CT, C-terminal; 1D, one-dimensional; ERK, extracellular-signal-regulated kinase; FBS, foetal bovine serum; GST, glutathione S-transferase; HRP, horseradish peroxidase; JX, juxtamembrane; LIF, leukaemia inhibitory factor; MysPDZ, myosin containing PDZ domain; NBCS, newborn calf serum; NP40, Nonidet P40; PY, phosphotyrosine; Shc, Src-homology collagen; SH2 domain, Src homology 2 domain; TBS, Tris-buffered saline.
Present address: Science, Technology & Innovation Section, Victorian Department of Innovation, Industry and Regional Development, Level 13, 55 Collins St., Melbourne 3000, Australia.
Present address: DNAX, 901 California Avenue, Palo Alto, CA 94304, U.S.A.