A critical intramolecular interaction for protein kinase Cepsilon translocation

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• Protein kinase C (PKC) activation requires translocation between cellular compartments and binding to RACKs.
• Inactive epsilonPKC is hypothesized to have its RACK-binding site intramolecularly engaged with a psiepsilonRACK sequence.
• An amino acid difference (aspartate in epsilonPKC vs. asparagine in epsilonRACK) distinguishes these interactions.

Purpose of the Study:

• To investigate the role of the intramolecular interaction between the psiepsilonRACK site and the RACK-binding site in epsilonPKC regulation.
• To determine how specific amino acid substitutions affect intramolecular interactions, RACK binding, and translocation.

Main Methods:

• Site-directed mutagenesis of epsilonPKC (Aspartate to Asparagine or Alanine).
• Assessing intramolecular interaction via proteolysis resistance.
• Monitoring hormone- or PMA-induced translocation in cells.
• In vitro binding assays with epsilonRACK.
• Mathematical modeling of translocation dynamics.

Main Results:

• Mutating Aspartate to Asparagine in epsilonPKC enhanced intramolecular interaction and slowed translocation.
• Substituting Aspartate with Alanine in epsilonPKC enabled RACK binding without activators and increased translocation rate.
• Mathematical modeling indicated translocation is a multi-step process.

Conclusions:

• Intramolecular interaction between the psiepsilonRACK site and RACK-binding site is crucial for epsilonPKC regulation.
• This interaction acts as a rate-limiting step in PKC translocation.
• Amino acid identity at this site dictates the balance between intramolecular interaction and RACK binding.