Photoaffinity labeling of a peptide substrate to myosin light chain kinase.
The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.
Gao
Z H
ZH
Department of Physiology, University of Texas Southwestern Medical Center at Dallas 75235, USA.
Zhi
G
G
Herring
B P
BP
Moomaw
C
C
Deogny
L
L
Slaughter
C A
CA
Stull
J T
JT
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000187:drug effects / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D010766:Phosphorylation; D010777:Photochemistry; D011817:Rabbits; D013379:Substrate Specificity
D000345:Affinity Labels; D002147:Calmodulin; D010455:Peptides; D010649:Phenylalanine; D000255:Adenosine Triphosphate; C053764:benzoylphenylalanine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium