KINETICS AND REGULATION OF 2 CATALYTIC SUBUNITS OF CAMP-DEPENDENT PROTEIN-KINASE FROM APLYSIA-CALIFORNICA

C(APL-A1) and C(APL-A2), two catalytic subunits of Aplysia cAMP-dependent protein kinase, are encoded by mRNAs generated by alternative splicing of transcripts of a gene that contains two mutually exclusive exon cassettes. The subunits are identical except for amino acids 142-183 of the 352 residues, which differ at 10 of 42 positions. C(APL-A1) and C(APL-A2) have now been expressed in insect cells and purified to homogeneity. The subunits differ in their catalytic properties, which have been determined with a series of synthetic peptide substrates. For example, k(cat) and K(m), values for the peptide LRRASLG (kemptide) are 42 s-1 and 36-mu-M and 28 s-1 and 17-mu-M for C(APL-A1) and C(APL-A2), respectively. C(APL-A1) and C(APL-A2) have different substrate specificities. For example, (k(cat)/K(m))peptide-T/(k(cat)/K(m))kemptide is 9.1 x 10(-3) for C(APL-A1) and 15 X 10(-3) for C(APL-A2), where peptide-T is the kemptide homologue LRRATLG. The subunits also differ in regulation as determined by their interactions with a purified type I regulatory subunit, which has an IC50 for C(APL-A1) that is 3.5 times higher than the IC50 for C(APL-A2). These modest differences reinforce accumulating evidence that the physiological state of a cell depends upon a spectrum of protein kinases with overlapping substrate specificities and regulatory properties.