FLS2 kinase activity is required for ciliary disassembly during which it undergoes dephosphorylation and inactivation.

Abstract

(A) FLS2 expression is not altered during ciliary disassembly. fls2 rescued cells expressing FLS2-HA was induced for ciliary disassembly by addition of NaPPi followed by immunoblotting with the indicated antibodies. (B) FLS2 undergoes gradual dephosphorylation during ciliary disassembly. A phos-tag immunoblotting was performed using antibodies as indicated. Steady state cells were treated with or without phosphatase (Ptase) to demonstrate that the gel mobility shift was caused by changes in protein phosphorylation. (C) Dephosphorylation of FLS2 results in loss of its kinase activity. fls2 cells expressing wild type FLS2-HA or kinase dead mutant K33R-HA were treated with 20 mM NaPPi for the indicated times. Wild type (WT) cells were used as a negative control. Cell lysates were incubated with anti-HA antibodies for immunoprecipitation followed by in vitro kinase assay. ATPγS was used as ATP donor and myelin basic protein as substrate. Anti-thiophosphate ester antibody was used to detect substrate phosphorylation. (D) The kinase activity of FLS2 is required for ciliary disassembly. Cells from WT, fls2 and kinase-dead mutant K33R were induced for ciliary disassembly by NaPPi treatment. Ciliary length was measured at the indicated times. Bars indicate SD.</p