The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements Gefitinib (Iressa) during development and tissue renewal. altered at Hdac11 casein kinase 2 and 1 consensus sites. In and mammalian cells. through a dual-kinase mechanism. RESULTS Identification of a major serine/threonine phospho-domain in α-catenin To identify phosphorylation sites in αE-catenin (αE-cat also known as catenin α-1) we affinity purified cadherin-free αE-cat- and β-catenin-containing complexes from human colon-cancer-derived SW480 cells and analyzed them with high mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray Gefitinib (Iressa) tandem mass spectrometry in collaboration with the Taplin facility (Harvard University or college Cambridge MA) (Fig.?1A-C). Four clustered phosphorylated serine (Ser S) and threonine (Thr T) residues were recognized that localize to a flexible linker region (amino acids 631-661) between the M-region and the C-terminal F-actin-binding domain name of αE-cat (Ishiyama et al. 2013 Rangarajan and Izard 2013 Yonemura et al. 2010 These sites were previously recognized in other large-scale phosphoproteomic screens where S641 is the most commonly observed site (mouse – Ballif et al. 2004 Huttlin et al. 2010 human – Beausoleil et al. 2004 Dephoure et al. 2008 Olsen et al. 2006 These sites appear to be responsible for most [32P]orthophosphate labeling of cellular αE-cat particularly S641 (Fig.?2M). Fig. 1. α-cat is a Gefitinib (Iressa) phosphoprotein. (A) Autoradiograph from SW480 cells labeled with [32P]orthophosphate and affinity precipitated (ppt) or immunoprecipitated (IP) with GST GST-ICAT or E-cad antibody. Nitrocellulose was first exposed to film ([ … Fig. 2. Identification of major CK2 and CK1 sites in α-cat. (A) Identification of S641 as the major CK2 site. Autoradiograph of [γ-32P]ATP kinase labeling of recombinant full-length (FL) and S641A (A alanine) αE-cat. The timecourse … Multiple sequence alignment of α-catenin proteins from diverse species indicates a conservation of Gefitinib (Iressa) the Ser and Thr residues in the linker between the M- and C-terminal (C)-domains (Fig.?1D E). Moreover phosphoproteomic screens in recognized seven possible phosphorylated Ser and Thr residues in this region including T645 which likely corresponds to S641 in αE-cat (Fig.?1E) (Zhai et al. 2008 Taken together these data support the identification of a major evolutionarily conserved phospho-domain in α-catenin proteins that we will refer to as the phospho-linker (P-linker) region. αE-cat is usually phosphorylated by a hierarchical dual-kinase mechanism Because mass spectrometry assigns phospho-modified residues imperfectly (Taus et al. 2011 (Fig.?1C) we sought to Gefitinib (Iressa) characterize αE-cat phosphorylation kinase assays revealed that S641 is the major CK2 site in αE-cat (Fig.?2A B; CK2 condition) whereas the Ser/Thr residues between 652 and 658 were the major CK1 sites (Fig.?2C D; CK1 condition). αE-cat phosphorylation by CK2 occurred rapidly (i.e. saturated within ～5?moments) in contrast to CK1 phosphorylation kinetics which were significantly slower (i.e. increased over 90?moments) (Fig.?2C D). As residues S652 S655 and T658 of αE-cat conform to a hierarchical CK1 phospho-scheme which prefers a negatively charged amino acid (aspartate or glutamate D or E) or phosphorylation at the ?3 position (Marin et al. 1994 Pulgar et al. 1999 we sought experimental evidence as to whether this plan Gefitinib (Iressa) also applies to αE-cat. We found that mutating the most N-terminal of the three consensus CK1 sites (S652A) reduced αE-cat phosphorylation by CK1 as effectively as removing all three CK1 sites (Fig.?2E F; 3A mutant). Moreover phosphoproteomic analysis of CK1-phosphorylated recombinant αE-cat detected a peptide with three phosphates (Fig.?1C) although the precise identification of the middle CK1 phosphorylation site could not be confidently assigned using the phosphoRS algorithm (Taus et al. 2011 Because CK1 phosphorylation techniques similar to that depicted in Fig.?2L are well described (Okamura et al. 2004 it is likely that S652 S655 and T658 conform to a hierarchical CK1-dependent phospho-scheme. Moreover evidence that αE-cat phosphorylation by CK1 increases over time whereas CK2 phosphorylation saturates within the first 5?moments (Fig.?2C D) further supports the sequential nature of CK1 phosphorylation of αE-cat..