Protein kinase C penetration into lipid bilayers

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Protein kinase C (PKC) plays a crucial role in cellular signaling pathways.
• Understanding PKC's interaction with cell membranes is vital for elucidating its function.

Purpose of the Study:

• To investigate the physical characteristics of protein kinase C's association and penetration into lipid bilayers.
• To determine the influence of lipid composition and environmental factors on PKC-bilayer interactions.

Main Methods:

• Utilized four distinct fluorescence probes to analyze PKC-lipid bilayer interactions.
• Employed carboxyfluorescein-filled unilamellar vesicles to assess bilayer permeability changes.
• Used N-methylpicolinium perchlorate as a hydrophilic quencher to probe enzyme microenvironments.
• Measured intrinsic tryptophan fluorescence quenching to determine the depth of enzyme penetration.

Main Results:

• PKC exhibited strong hydrophobic and electrostatic interactions with lipid bilayers, increasing vesicle permeability.
• The extent of PKC-bilayer interaction was concentration-dependent on phosphatidylserine.
• Tryptophan residues involved in conformational changes were located in hydrophobic regions of PKC.
• PKC penetrated the lipid bilayer to the C-16 position of fatty acid probes.
• Divalent cations did not affect PKC association or penetration and had no significant impact on substrate phosphorylation.

Conclusions:

• PKC associates with and penetrates lipid bilayers via hydrophobic and electrostatic mechanisms.
• Phosphatidylserine concentration modulates the strength of PKC-bilayer interactions.
• Enzyme penetration into the bilayer is substantial and independent of divalent cations, suggesting a specific membrane-binding mode.