Characterization of the interaction of phorbol esters with the C1 domain of MRCK (myotonic dystrophy kinase-related Cdc42 binding kinase) alpha/beta.
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.
Choi
Sung Hee
SH
Laboratory of Cancer Biology and Genetics, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Czifra
Gabriella
G
Kedei
Noemi
N
Lewin
Nancy E
NE
Lazar
Jozsef
J
Pu
Yongmei
Y
Marquez
Victor E
VE
Blumberg
Peter M
PM
D000595:Amino Acid Sequence; D001692:Biological Transport; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D065636:Myotonin-Protein Kinase; D010703:Phorbol Esters / Q000737:chemistry*; D010718:Phosphatidylserines / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000502:physiology*
D010703:Phorbol Esters; D010718:Phosphatidylserines; C105518:CDC42BPA protein, human; C118666:CDC42BPB protein, human; D011505:Protein-Tyrosine Kinases; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases