Fig 5. In vivo, CK1δ T347 is phosphorylated in trans.

A) Characterization of a specific CK1δ pT347 phospho-epitope antibody. HEK293 cells over-expressing wild-type or T347A 6Myc-CK1δ were treated with 40 nM calyculin A for 30 minutes prior to lysis and immunoblotting with anti-pT347 or anti-CK1δ antibodies. Arrows indicate Myc-tagged CK1δ while asterisks indicate endogenous CK1δ, which also serves as a load control. B) Phosphorylation of T347 on endogenous CK1δ is reversed by alkaline phosphatase. Endogenous CK1δ was immunoprecipitated from calyculin A-treated HEK293 cells. Pelleted beads were treated with either vehicle or alkaline phosphatase (ALP) prior to analysis by SDS-PAGE and immunoblotting with the indicated antibodies. Calyculin-enhanced CK1δ phosphorylation causes a marked electrophoretic mobility shift that is reversed by alkaline phosphatase. Closed circle denotes the IgG heavy chain present in the immunoprecipitate. WCE denotes whole cell extract. C) T347 is not a CK1δ autophosphorylation site. Wild-type or catalytically-inactive K38A 6Myc-CK1δ were transiently expressed in HEK293 cells. Following a 30 minute treatment with either DMSO or 40 nM calyculin A, cells were lysed and immunoblotted for anti-pT347 or anti-CK1δ antibodies. Kinase-dead CK1δ has a much reduced calyculin-induced electrophoretic mobility shift but is still phosphorylated on T347. Myc-tagged CK1δ indicated by curly brackets. Open circle denotes a Calyculin A dependent non-specific band. D) Phosphorylation of CK1δ at T347 is not dependent on CK1 activity. HEK293 cells over-expressing wild-type CK1δ were treated with CK1 inhibitors PF670462 (PF670, 1 μM), PF4800567 (PF480, 1 μM), or D4476 (30 μM) for 3 hours, followed by addition for 30 min of either DMSO or 40 nM Calyculin A. Cell lysates were immunoblotted for anti-pT347 or anti-Myc antibodies. CK1 inhibitors block the autophosphorylation-induced mobility shift but do not decrease phosphorylation of T347. Myc-tagged CK1δ indicated by curly brackets.