Abstract
Protein kinase C-related kinase 1 (PRK1) or PKN is a protease and lipid activated protein kinase that acted downstream of the RhoA or Rac1 pathway. PRK1 comprises a unique regulatory domain and a PKC homologous kinase domain. The regulatory domain of PRK1 consists of homologous region -1 (HR1) and -2 (HR2). PRK1-(HR1) features a pseudosubstrate motif that overlapped with the putative cardiolipin and known RhoA binding sites. In fact, cardiolipin is the most potent lipid activator for PRK1 in respect of its either auto- or substrate phosphorylation activity. This study was thus aimed to characterize the binding region(s) of cardiolipin that was previously suggested for the regulatory domain of PRK1. The principal findings of this work established (i) PRK1-(HR1) folded into an active conformation where high affinity binding sites (mainly located in HR1a subdomain) were accessible for cardiolipin binding to protect against limited Lys-C digestion, (ii) the binding nature between acidic phospholipids and PRK1 (HR1) involved both polar and nonpolar components consistent with the amphipathic nature of the known cardiolipin-binding motifs, (iii) identification of the molecule masses of the Lys-C fragments of PRK1-(HR1) complexed with cardiolipin molecule, and (iv) appreciable reductions in the secondary structural contents at 222 nm measured by circular dichroism analyses demonstrated the binding of cardiolipin elicited the disruptive effect that was most evident among all phospholipids tested, suggestive of a functional correlation between the extents of helical disruption and PRK1 activation.