In vitro assay and characterization of the farnesylation-dependent prelamin A endoprotease.
The 72-kDa nuclear lamina protein lamin A is synthesized as a 74-kDa farnesylated precursor. Conversion of this precursor to mature lamin A appears to be mediated by a specific endoprotease. Prior studies of overexpressed wild-type and mutant lamin A proteins in cultured cells have indicated that the precursor possesses the typical carboxyl-terminal S-farnesylated, cysteine methyl ester and that farnesylation is required for endoproteolysis to occur. In this report, we describe the synthesis of an S-farnesyl, cysteinyl methyl ester peptide corresponding to the carboxyl-terminal 18 amino acid residues of human prelamin A. This peptide acts as a substrate for the prelamin A endoprotease in vitro, with cleavage of the synthetic peptide at the expected site between Tyr657 and Leu658. Endoproteolytic cleavage requires the S-prenylated cysteine methyl ester and, in agreement with transfection studies, is more active with the farnesylated than geranylgeranylated cysteinyl substrate. N-Acetyl farnesyl methyl cysteine is shown to be a noncompetitive inhibitor of the enzyme. Taken together, these observations suggest that there is a specific farnesyl binding site on the enzyme which is not at the active site.
Kilic
F
F
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614-0581, USA.
Dalton
M B
MB
Burrell
S K
SK
Mayer
J P
JP
Patterson
S D
SD
Sinensky
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D034904:Lamin Type A; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*
D034904:Lamin Type A; D009687:Nuclear Proteins; D011498:Protein Precursors; C090764:prelamin A; C057785:S-farnesylcysteine; D010450:Endopeptidases; C104823:prelamin A endoprotease; D003545:Cysteine