Taxol and lipopolysaccharide activation of a murine macrophage cell line and induction of similar tyrosine phosphoproteins.
Taxol, a unique antimitotic drug, is thought to exert its antitumor activity by binding to and promoting the assembly of microtubules. Studies on the mechanism of action of Taxol have focused mainly on this ability to induce microtubule polymerization. Recent evidence suggests that Taxol affects novel intracellular targets within macrophages and neutrophils. To investigate further the mechanism of action of Taxol on macrophages, we have examined the pattern of tyrosine protein phosphorylation, using antiphosphotyrosine monoclonal antibodies (MAbs) in a RAW 264.7 (RAW) macrophage cell line. We found that Taxol, like lipopolysaccharides (LPS), caused a marked increase in tyrosine phosphorylation of three proteins having M(r) of 40 (p40), 41 (p41), and 43 (p43) kd in RAW cells. Immunoprecipitation of these tyrosine phosphoproteins followed by Western blotting with a microtubule-associated protein-2 (MAP-2) kinase MAb revealed that both Taxol and LPS induced the tyrosine phosphorylation of a MAP-2 kinase-like protein. In addition, MAP-2 kinase-like activity was stimulated in the presence of Taxol or LPS. Examination of cellular mRNA levels in LPS and Taxol-activated macrophages by Northern blot analysis revealed increased expression of Interleukin-1 beta, and tumor necrosis factor-alpha cytokine mRNAs. Because Taxol promotes tubulin assembly, we examined the effect of LPS on microtubule polymerization. LPS had no polymerizing activity over Taxol alone. We conclude that Taxol and LPS have a common target in macrophages that is a critical component of the signal transduction pathway that mediates LPS cellular responses.
Carboni
J M
JM
Department of Experimental Therapeutics, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, N.J. 08543.
Singh
C
C
Tepper
M A
MA
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation / Q000187:drug effects*; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D010750:Phosphoproteins / Q000096:biosynthesis*; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism*
D016207:Cytokines; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D014443:Tyrosine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017239:Paclitaxel