Allosteric Regulation of Protein Kinase PKCζ by the N-Terminal C1 Domain and Small Compounds to the PIF-Pocket

Abstract

Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by the N-terminal region cannot be explained by a simple pseudosubstrate inhibitory mechanism. Notably, we found that the C1 domain allosterically inhibits PKCζ activity and verified an allosteric communication between the PIF-pocket of atypical PKCs and the binding site of the C1 domain. Finally, we developed low-molecular-weight compounds that bind to the PIF-pocket and allosterically inhibit PKCζ activity. This work establishes a central role for the PIF-pocket on the regulation of PKCζ and allows us to envisage development of drugs targeting the PIF-pocket that can either activate or inhibit AGC kinases.

Partitioning-defective Protein 6 (Par-6) Activates Atypical Protein Kinase C (aPKC) by Pseudosubstrate Displacement

“…These values are similar to those from other catalytic domains from the AGC family of kinases (31,32). The dramatic difference between the activity of full-length aPKC and its isolated kinase domain suggests that aPKC is autoinhibited and does not require other elements for regulation.…”

“…Briefly, the purified aPKC variants and aPKC/Par-6 complex were diluted to concentrations at which the incorporation of radiolabeled phosphate from [␥- 32 P]ATP into MBP-Lgl peptide was linear with respect to time and the enzyme concentrations. The diluted enzymes were preincubated in the assay buffer (50 mM TrisHCl, pH 7.5, 100 mM NaCl, 10 mM MgCl 2 ) with a wide range of MBP-Lgl peptide concentrations at 30°C for 5 min.…”

25 Years of Small Molecular Weight Kinase Inhibitors: Potentials and Limitations

“…Targeting the allosteric sites on protein kinases may also lead to the identification of activators rather than inhibitors, which could be useful for therapeutic intervention or as pharmacological tools. In particular, compounds targeting the PIF pocket (the hydrophobic motif present in the N-terminal lobe of AGC kinases) of either PDK1 or PKCz can either act as activators (Hindie et al, 2009) or substrate-selective inhibitors (Lopez-Garcia et al, 2011;Sadowsky et al, 2011;Busschots et al, 2012). Similarly, the myr-pocket binders of ABL can be converted into activators if they are designed not to bend helix I of the ABL kinase domain Yang et al, 2011).…”

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

“…For instance, to measure the strength of an intramolecular interaction between two domains it is typical to split them into two individual peptides (11). An alternative approach is to truncate or mutagenize a proposed interaction interface and look at a functional consequence associated with the loss of the interaction (12)(13)(14). In the first approach the strength of an interaction can be underestimated given that when the two interacting domains are in the same polypeptide, they will likely have a high local concentration that can affect the on-rate of the interaction.…”