Expression of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli: multiple isozymes reflect different phosphorylation states.
The catalytic subunit of mouse cAMP-dependent protein kinase expressed in Escherichia coli was separated into three distinct species using Mono-S ion exchange chromatography. These isoenzymes corresponded to three isoelectric variants with pIs of 6.4 (30%), 7.2 (60%) and 8.2 (10%). The Stokes' radius of each form was 27.7, 27.1 and 26.3 A respectively. Using electrospray mass spectroscopy the differences between the isozymes were shown to be due to phosphorylation, with each form differing by 80 mass units corresponding to a single phosphate. The fully phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three. Since the enzyme is not phosphorylated when active site mutations are introduced into the C-subunit, these phosphates are incorporated in an autocatalytic mechanism and are not due to E. coli protein kinases. When the recombinant enzyme was compared with the mammalian porcine heart enzyme significant differences in post-translational modifications were observed. The mammalian enzyme could also be separated into two isozymes. However, in contrast to the recombinant enzyme, the mammalian isozymes displayed an identical mass of 40 840. This correlated with two different post-translational modifications: two phosphates and an N-terminal myristyl moiety. The importance of post-translational modifications, and in particular the phosphorylation state, for the expression of eukaryotic proteins in E. coli is discussed.
Herberg
F W
FW
Department of Chemistry, University of California at San Diego, La Jolla 92093-0654.
Bell
S M
SM
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D007526:Isoelectric Point; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D009206:Myocardium / Q000201:enzymology; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
D007527:Isoenzymes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases