PHOSPHORYLATION OF T-558 OF MOESIN DETECTED BY SITE-SPECIFIC ANTIBODIES IN RAW264.7 MACROPHAGES

To determine, whether (558)Thr in the carboxyl-terminal domain of moes in is phosphorylated in cells other than platelets, rabbit phosphoryla tion state-specific antibodies were made to the chemically phosphoryla ted synthetic hexapeptide KYKpTLR of the moesin sequence, as well as t o the unphosphorylated form. The affinity-purified antibody population s were specific for either the phosphorylated or the unmodified peptid e conjugated to BSA. Site-specific phosphorylation of moesin is detect ed in RAW macrophages by Western blot analysis, and immunofluorescence studies demonstrate that phosphorylated moesin is localized in filopo dial protrusions. After pretreatment with the phosphatase inhibitor ca lyculin A, a similar effect to that seen in platelets is found, namely a substantial increase in moesin phosphorylation at (558)Thr and redi stribution of phospho-moesin together with F-actin into one or more ri ng-like structures in the cytoplasm, presumably due to binding of phos phorylated moesin to F-actin. (C) 1996 Academic Press, Inc.