# Lipid-Binding Regions within PKC-Related Serine/Threonine Protein Kinase N1 (PKN1) Required for Its Regulation

## TL;DR

This paper identifies specific lipid-binding regions in the PKN1 kinase, which could help design inhibitors to block its membrane signaling.

## Contribution

The study reveals new lipid-binding motifs and structural conformations in the C2 domain of PKN1.

## Key findings

- PKN1-(C2) contains conserved amphipathic cardiolipin-binding motifs.
- Trimeric PKN1-(C2) adopts a β-stranded conformation.
- Two types of cardiolipin binding are identified, with hydrophobic interactions being key at high salt.

## Abstract

PKC-related serine/threonine
protein kinase N1 (PKN1)
is a protease/lipid-activated
protein kinase that acts downstream of the RhoA and Rac1 pathways.
PKN1 comprises unique regulatory, hinge region, and PKC homologous
catalytic domains. The regulatory domain harbors two homologous regions,
i.e., HR1 and C2-like. HR1 consists of three heptad repeats (HR1a,
HR1b, and HR1c), with PKN1-(HR1a) hosting an amphipathic high-affinity
cardiolipin-binding site for phospholipid interactions. Cardiolipin
and C18:1 oleic acid are the most potent lipid activators of PKN1.
PKN1-(C2) contains a pseudosubstrate sequence overlapping that of
C20:4 arachidonic acid. However, the cardiolipin-binding site(s) within
PKN1-(C2) and the respective binding properties remain unclear. Herein,
we reveal (i) that the primary PKN1-(C2) sequence contains conserved
amphipathic cardiolipin-binding motif(s); (ii) that trimeric PKN1-(C2)
predominantly adopts a β-stranded conformation; (iii) that two
distinct types of cardiolipin (or phosphatidic acid) binding occur,
with the hydrophobic component playing a key role at higher salt levels;
(iv) the multiplicity of C18 fatty acid binding to PKN1-(C2); and
(v) the relevance of our lipid-binding parameters for PKN1-(C2) in
terms of kinetic parameters previously determined for the full-length
PKN1 enzyme. Thus, our discoveries create opportunities to design
specific mammalian cell inhibitors that disrupt the localization of
membrane-associated PKN1 signaling molecules.