Abstract
C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP. MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase), a member of the dystrophia myotonica protein kinase family that functions downstream of Cdc42, contains a C1 domain with substantial homology to that of the diacylglycerol/phorbol ester-responsive C1 domains and has been reported to bind phorbol ester. We have characterized here the interaction of the C1 domains of the two MRCK isoforms alpha and beta with phorbol ester. The MRCK C1 domains bind [20-(3)H]phorbol 12,13-dibutyrate with K(d) values of 10 and 17 nm, respectively, reflecting 60-90-fold weaker affinity compared with the protein kinase C delta C1b domain. In contrast to binding by the C1b domain of PKCdelta, the binding by the C1 domains of MRCK alpha and beta was fully dependent on the presence of phosphatidylserine. Comparison of ligand binding selectivity showed resemblance to that by the C1b domain of PKCalpha and marked contrast to that of the C1b domain of PKCdelta. In intact cells, as in the binding assays, the MRCK C1 domains required 50-100-fold higher concentrations of phorbol ester for induction of membrane translocation. We conclude that additional structural elements within the MRCK structure are necessary if the C1 domains of MRCK are to respond to phorbol ester at concentrations comparable with those that modulate PKC.
Highlights
C1 domains mediate the recognition and subsequent signaling response to diacylglycerol and phorbol esters by protein kinase C (PKC) and by several other families of signal-transducing proteins such as the chimerins or RasGRP
The isolated C1 domains of MRCK ␣ and  were expressed as glutathione S-transferase (GST) fusion proteins in E. coli and partially purified as described under “Experimental Procedures.” [3H]PDBu binding was measured in the presence of 100 g/ml of phosphatidylserine
Comparison of the Redistribution of the C1 Domain of MRCK␣ and the C1b Domain of PKC␦ in LNCaP Cells after Phorbol Ester Treatment—Using the GFP fusion protein with the C1 domain of MRCK␣, we examined its subcellular distribution in living LNCaP cells as a function of time after treatment with various doses of phorbol 12-myristate 13-acetate (PMA) and compared its response with that of the C1b domain of PKC␦
Summary
Materials—[20-3H]Phorbol 12,13-dibutyrate ([3H]PDBu) (17 Ci/mmol) was purchased from PerkinElmer. 1,2-Dioctanoyl glycerol was purchased from Sigma. Antimouse IgG was purchased from Bio-Rad. Construction of GST- and GFP-fused C1 Domains of MRCK␣ and MRCK—The C1 domains of MRCK ␣ and  were generated by PCR using the Platinum Pfx DNA polymerase (Invitrogen). Expression and Imaging of GFP-tagged C1 Domains in Living Cells—LNCaP cells (obtained from ATCC, Manassas, VA) were cultured at 37 °C in RPMI 1640 containing 10% fetal bovine serum, penicillin (50 units/ml), and streptomycin (0.05 mg/ml) in a 5% CO2 atmosphere. The plasmid DNAs of the GFP-fused individual C1 domain of MRCK␣ and the C1b domain of PKC␦ were transfected into LNCaP cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The cells were washed with ice-cold Dulbecco’s phosphate-buffered saline (KD Medical, Inc., Columbia, MD) and harvested with 120 l of 50 mM Tris-HCl, pH 7.4, plus protease inhibitor mixture (Roche Applied Science). In a typical membrane translocation assay, ten concentrations of ligand were used
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