Abstract
The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCdelta by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCdelta have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCdelta and the origin of its specific subcellular localization in response to DAG.
Highlights
Protein kinase C (PKC)1 are a family of serine/threonine kinases that mediate a wide variety of cellular processes [1, 2]
The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins
In vitro membrane binding studies of individual membrane targeting domains (C1A, C1B, and C2) of PKC␦ and the full-length PKC␦ and mutants by isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and monolayer penetration analyses, as well as cellular membrane translocation measurements, reveal that PKC␦ has a unique membrane binding and activation mechanism, which derives from the distinct DAG affinities of the two C1 domains and the unique conformational properties of this novel PKC isoform
Summary
Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoylsn-glycero-3-phosphoinositol (POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoserine (POPS), cardiolipin, sn-1,2-dioctanoylglycerol (DiC8), and sn-1,2-dioleoylglycerol (DiC18) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Determination of PKC Activity—Activity of PKC␦ was assayed by measuring the initial rate of [32P]phosphate incorporation from [␥-32P]ATP (50 M, 0.6 Ci/tube) into myelin basic protein (200 g/ml) or protamine sulfate (200 g/ml) (both from Sigma). The second flow cell contained the surface coated with vesicles with varying lipid compositions (e.g. POPC/POPS/DiC18 ϭ 59:40:1) at 4500 resonance units. The transfection medium was removed, the cells were washed once with fetal bovine serum-supplemented DMEM, and overlaid with fetal bovine serum-supplemented DMEM containing Zeocin and 5 g/ml ponasterone A to induce protein production for 16 –24 h. The translocation of protein and subcellular localization of lipid was simultaneously monitored at fixed intervals (every 20 s) after 150 l of HEK buffer containing 0.1 mg/l OPG (or DiC8) was added. Note that each experiment was repeated at least three times on a given day and was repeated at least two different days with different transfected cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
