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 M ono-S ion exchange chromatography. These isozymes corresponded to thre e 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 angstrom resp ectively. 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 fu lly phosphorylated recombinant enzyme contained four phosphates while the dominant isozyme contained only three. Since the enzyme is not pho sphorylated when active site mutations are introduced into the C-subun it, these phosphates are incorporated in an autocatalytic mechanism an d are not due to E.coli protein kinases. When the recombinant enzyme w as compared with the mammalian porcine heart enzyme significant differ ences in post-translational modifications were observed. The mammalian enzyme could also be separated into two isozymes. However, in contras t to the recombinant enzyme, the mammalian isozymes displayed an ident ical mass of 40 840. This correlated with two different post-translati onal 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 i n E.coli is discussed.