Fig. 1: JAK2 phosphorylates CARM1 in vitro.
From: Tyrosine phosphorylation of CARM1 promotes its enzymatic activity and alters its target specificity
A JAK2 phosphorylates CARM1 in an in vitro kinase assay, in which active JAK2 kinase and recombinant CARM1 proteins were used; PAK1 was used as a positive control. The amounts of protein in the reaction are indicated. The phosphorylation of CARM1 was completely abolished by the JAK1/JAK2 inhibitor (ruxolitinib, RUX; 500 nM) (lanes 7 and 8). The experiment was repeated independently twice. B Peptide fragments in the mass spectrometry analysis were generated from proteolytic cleavage of CARM1 following in vitro kinase assays in the presence of active JAK2 kinase. Tyrosine-149 (Y149) along with the series of y- and b-ions, including the phosphorylated residue, is shown as the phosphorylated peptide (EESSAVQpYF). C Peptide fragments in the mass spectrometry analysis were generated from proteolytic cleavage of CARM1 following in vitro kinase assays in the absence of active JAK2 kinase. The peptide fragment around Y149 residue (EESSAVQYF) is shown without phosphorylation of tyrosine, indicating no gain in molecular weight of 80 Da (i.e. the weight of PO4). D Peptide fragments in the mass spectrometry analysis were generated from proteolytic cleavage of CARM1 following in vitro kinase assays in the presence of active JAK2 kinase. Tyrosine-334 (Y334) along with the series of y- and b-ions, including the phosphorylated residue, is shown as the phosphorylated peptide (GAAVDEpYFR). (E) Peptide fragments in the mass spectrometry analysis were generated from proteolytic cleavage of CARM1 following in vitro kinase assays in the absence of active JAK2 kinase. The peptide fragment around Y334 residue (GAAVDEYFR) is shown without phosphorylation of tyrosine, indicating no gain in molecular weight of 80 Da. F The regions containing amino acid residues Y149 and Y334 are located within the core catalytic domain (residue 140-480) of CARM1. Residues 28-140 in CARM1 are highly homologous to a family of Drosophila-enbaled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domains, which specifically bind to target proline-rich sequences with low affinity and high specificity.
