IDENTIFICATION OF CRITICAL DETERMINANTS FOR AUTOINHIBITION IN THE PSEUDOSUBSTRATE REGION OF TYPE-I-ALPHA CAMP-DEPENDENT PROTEIN-KINASE

The consensus substrate site for cAMP-dependent protein kinase (PKA) i s Arg-Arg-Xaa-Ser(P)-Xaa and the autoinhibitory domain of the PKA type I alpha regulatory subunit (RI subunit) contains a similar sequence, Arg(92)-Arg-Arg-Arg-Gly-Ala-Ile-Ser-Ala-Glu. The italicized amino acid s form a putative pseudosubstrate site (Ser is replaced with Ala), whi ch together with adjacent residues could competitively inhibit substra te phosphorylation by the PHA catalytic subunit (C subunit). The prese nt studies determine the contributions of Arg(92-95), Ile(98), and Glu (101) to inhibitory potency. Amino-terminal truncation of RI subunit t hrough Arg(92) (Delta 1-92) or Arg(93) (Delta 1-93) had no detectable effect on inhibition of C subunit. Truncation through Arg(94) (Delta 1 -94), or point mutation of Arg(95) within truncated mutants (Delta 1-9 3.R95A or Delta 1-92.R95A), caused a dramatic reduction in inhibitory potency. Truncation through Arg(95) (Delta 1-95) had a greater effect than did replacement or deletion of Arg(94) or Arg(95) alone. Using fu ll-length RI subunit, the inhibitory potency was reduced by replacing Ile(98) with Ala, Gly, or Gln, but not by replacing it with Val. The i nhibitory potency of RI subunit was unchanged when Glu(101) was replac ed with Ala or Gln. It is concluded that Arg(94), Arg(95) and, to a le sser extent, Ile(98) are vital constituents of PKA autoinhibition by t ype I alpha R subunit.