MYOSIN LIGHT-CHAIN PHOSPHORYLATION DOES NOT INCREASE DURING YEAST PHAGOCYTOSIS BY MACROPHAGES

We have studied the role of myosin II light chain phosphorylation in y east 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. Immunop recipitation of myosin II from cells labeled with P-32, using an affin ity-purified antibody to myosin II purified from J774 cells, demonstra ted phosphorylation of both the myosin heavy chain and the 20-kDa ligh t chain (PMLC) prior to the addition of the opsonized yeast. However, the levels of heavy chain and PMLC phosphorylation did not change duri ng the linear phase of yeast uptake by J774 cells. Other experiments d emonstrated that the amount of myosin II associated with the cytoskele ton did not change during phagocytosis, further supporting the observa tion 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 greate r detail the role of myosin II phosphorylation in phagocytosis. First, antibodies to myosin light chain kinase (MLCK), the enzyme that phosp horylates PMLC, were electroinjected into J774 cells. These antibodies , which inhibit MLCK activity, inhibited chemotaxis as previously desc ribed but had no effect on phagocytosis. Second, quantitation of phago cytosis and chemotaxis following treatment with the phosphoprotein pho sphatase inhibitor okadaic acid demonstrated that chemotaxis was much more sensitive than phagocytosis to okadaic acid treatment; at 0.3 muM okadaic acid, there is a substantial increase in myosin phosphorylati on and chemotaxis is inhibited by 60%, whereas phagocytosis is unaffec ted. 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 r espect to mediating energy-dependent cellular processes in macrophages .