Regulation of polymorphonuclear leukocyte phagocytosis by myosin light chain kinase after activation of mitogen-activated protein kinase

Polymorphonuclear leukocyte (PMNL) phagocytosis mediated by Fc gamma RII pr oceeds in concert with activation of the mitogen-activated protein (MAP) ki nase, extracellular signal-regulated kinase ERK2, We hypothesized that myos in light chain kinase (MLCK) could be phosphorylated and activated by ERK, thereby linking the MAP kinase pathway to the activation of cytoskeletal co mponents required for pseudopod formation. To explore this potential linkag e, PMNLs were challenged with antibody-coated erythrocytes (EIgG). Peak MLC K activity, 3-fold increased over controls, occurred at 4 to 6 minutes, cor responding with the peak rate of target ingestion and ERK2 activity. The ML CK inhibitor ML-7 (10 mu mol/L) inhibited both phagocytosis and MLCK activi ty to basal values, thereby providing further support for the linkage betwe en the functional response and the requirement for MLCK activation. The MAP K kinase (MEK) inhibitor PD098059 inhibited phagocytosis, MLCK activity, an d ERK2 activity by 80% to 90%, To directly link ERK activation to MLCK acti vation, ERK2 was immunoprecipitated from PMNLs after EIgG ingestion. The is olated ERK2 was incubated with PMNL cytosol as a source of unactivated MLCK and with MLCK substrate; under these conditions ERK2 activated MLCK, resul ting in phosphorylation of the MLCK substrate or of the myosin light chain itself. Because MLCK activates myosin, we evaluated the effect of directly inhibiting myosin adenosine triphosphatase using 2,3-butanedione monoxime ( BDM) and found that phagocytosis was inhibited by more than 90% but MLCK ac tivity remained unaffected. These results are consistent with the interpret ation that MEK activates ERK, ERK2 then activates MLCK, and MLCK activates myosin, MLCK activation is a critical step in the cytoskeletal changes resu lting in pseudopod formation, (Blood, 2000;95:2407-2412) (C) 2000 by The Am erican Society of Hematology.