Inactive membrane protein kinase Cs: a possible target for receptor signalling

Area of Science:

• Cell Biology
• Biochemistry
• Enzymology

Background:

• Protein kinase Cs (PKCs) are key cell signaling enzymes.
• PKC activation is typically associated with translocation from cytosol to membranes.
• Emerging evidence suggests alternative activation mechanisms independent of translocation.

Purpose of the Study:

• To investigate the presence and activation of PKCs resident in native cell membranes.
• To determine if extracellular stimuli can activate membrane-bound PKCs without cytosolic enzyme translocation.
• To explore the role of membrane-resident PKCs in cellular responses to growth factors and signaling molecules.

Main Methods:

• Developed a PKC assay to measure enzyme activity directly in isolated native membranes.
• Stimulated various cell lines (3T3-L1, CTLL-2, WEHI-231, S49) with growth factors (EGF, FGF, IL-2).
• Administered diacylglycerols (OAG) and phorbol esters to intact cells to assess PKC activation and translocation.

Main Results:

• Identified an activatable pool of PKCs within native cell membranes across different cell types.
• Growth factors (EGF, FGF, IL-2) stimulated membrane PKC activity without significant cytosolic translocation.
• Low concentrations of OAG and phorbol esters activated membrane PKCs more readily than they induced cytosolic translocation.
• Phosphatidylcholine-specific phospholipase C selectively activated membrane PKCs.

Conclusions:

• Cell membranes harbor a pool of activatable PKCs.
• These membrane-resident PKCs respond to lower levels of extracellular stimuli compared to cytosolic PKCs.
• Activation can occur via signals generating diacylglycerols from non-polyphosphoinositide phospholipids.