Analysis of PKD1 autophosphorylation in vitro and confirmation of stoichiometric S742 phosphorylation of a recombinant PRKD1 kinase domain

Protein kinase D1 (PRKD1, also referred to as PKD1), has been proposed to undergo autophosphorylation on serine 742 and potentially also on serine 738 in the activation loop of its kinase domain. We used recombinant PKD1 kinase domain to test whether PKD1 is indeed able to undergo autophosphorylation of its kinase domain and how many sites could be phosphorylated. In order to obtain fully phosphorylated PKD1 kinase domain we adapted our autophosphorylation protocol to obtain monophosphorylated PKD1 kinase domain and determined the phosphorylation site by ArgC digest and phosphopeptide mapping.