Myosin light chain phosphorylation does not increase during yeast phagocytosis by macrophages.
We have studied the role of myosin II light chain phosphorylation in yeast phagocytosis by J774 cells. J774 cells, which are mouse cells of monocyte/macrophage lineage, ingest opsonized yeast particles, and the rate of internalization is linear for 60 min at 37 degrees C. Immunoprecipitation of myosin II from cells labeled with 32P, using an affinity-purified antibody to myosin II purified from J774 cells, demonstrated phosphorylation of both the myosin heavy chain and the 20-kDa light chain (PMLC) prior to the addition of the opsonized yeast. However, the levels of heavy chain and PMLC phosphorylation did not change during the linear phase of yeast uptake by J774 cells. Other experiments demonstrated that the amount of myosin II associated with the cytoskeleton did not change during phagocytosis, further supporting the observation that PMLC phosphorylation does not increase during phagocytosis. In contrast, F-actin increased by 1.6-fold during the linear phase of phagocytosis. Two additional approaches were used to analyze in greater detail the role of myosin II phosphorylation in phagocytosis. First, antibodies to myosin light chain kinase (MLCK), the enzyme that phosphorylates PMLC, were electroinjected into J774 cells. These antibodies, which inhibit MLCK activity, inhibited chemotaxis as previously described but had no effect on phagocytosis. Second, quantitation of phagocytosis and chemotaxis following treatment with the phosphoprotein phosphatase inhibitor okadaic acid demonstrated that chemotaxis was much more sensitive than phagocytosis to okadaic acid treatment; at 0.3 microM okadaic acid, there is a substantial increase in myosin phosphorylation and chemotaxis is inhibited by 60%, whereas phagocytosis is unaffected. These data indicate that PMLC phosphorylation and, by implication, myosin II are not involved in yeast phagocytosis. They also suggest that PMLC phosphorylation displays a high degree of specificity with respect to mediating energy-dependent cellular processes in macrophages.
de Lanerolle
P
P
Department of Physiology and Biophysics, University of Illinois, Chicago 60612.
Gorgas
G
G
Li
X
X
Schluns
K
K
D000818:Animals; D002460:Cell Line; D004988:Ethers, Cyclic / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology*; D051379:Mice; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D019319:Okadaic Acid; D010587:Phagocytosis; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000276:immunology*
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D009219:Myosin-Light-Chain Kinase; D009218:Myosins