Kinases and phosphatases involved in early Colony Stimulating Factor-1 signal transduction in the macrophage

Abstract

Colony Stimulating Factor-1 (CSF-1) transduces mitogenic and differentiation signals in mononuclear phagocytes by binding and activating its cognate receptor tyrosine kinase, causing the rapid tyrosine phosphorylation of the receptor and many other cellular proteins. The effect of rapid CSF-1 stimulation on any kinases and phosphatases that are tyrosine phosphorylated or associate with tyrosine phosphorylated proteins in the anti-phosphotyrosyl reactive fraction (YPRF) of macrophages was investigated.;YPRF proteins were purified from BAC1.2F5 macrophages stimulated with CSF-1 for 120min. at 4{dollar}\sp\circ{dollar}C, conditions equivalent to a rapid 30sec. stimulation at 37{dollar}\sp\circ{dollar}C that maximize the yield of tyrosine phosphorylated proteins, and separated into cytosolic (cytYPRF) and membrane (memYPRF) fractions which were assayed for phosphotransferase activity. Upon CSF-1 stimulation, cytYPRF and memYPRF serine/threonine kinase activity decreased by {dollar}\sim{dollar}40% and {dollar}\sim{dollar}60% respectively. Protein kinase A whose protein concentration remained unchanged contributed {dollar}\sim{dollar}90% of the regulated cytYPRF activity, and {dollar}\sim{dollar}40% of the memYPRF activity. CSF-1 also recruited the delta isoform of protein kinase C (PKC-{dollar}\delta{dollar}) to the memYPRF, but its activity could not be detected.;CSF-1 increased memYPRF tyrosine kinase activity 6-fold of which {dollar}\sim{dollar}90% was contributed by the CSF-1 receptor. CytYPRF activity was unaffected by CSF-1.;CSF-1 increased cytYPRF tyrosine phosphatase (PTPase) activity 5-fold. Ninety percent was contributed by protein tyrosine phosphatase-1C (PTP1C) and PTP1D was a minor component. CSF-1 induced the tyrosine phosphorylation of PTP1C, resulting in a 6-fold increase in its specific activity associated with enhanced substrate affinity. CSF-1 induced a 10-fold increase in the memYPRF PTPase activity of which PTP1C comprised {dollar}\sim{dollar}40% and RPTP{dollar}\alpha{dollar}, LAR, and the novel PTP{dollar}\phi{dollar} contributed to the remainder ({dollar}\sim{dollar}10%). PTP1D was also recruited to the memYPRF by CSF-1 but without measurable activity. PTP{dollar}\phi{dollar} and PTP1C were further characterized by investigating the kinetics and stability of each enzyme. No serine phosphatase activity was detected in the YPRF.;Thus, early events in CSF-1 signaling involve activation of the CSF-1 receptor, inhibition of PKA in the cytYPRF, and the recruitment of PKC-{dollar}\delta{dollar} and several PTPases to the YPRF including PTP1C, PTP1D, RPTP{dollar}\alpha{dollar}, LAR, and PTP{dollar}\phi{dollar}. The implications of these results are discussed.