Two well-defined motifs in the cAMP-dependermt protein kinase inhibitor (PKI alpha) correlate with inhibitory and nuclear export function

The heat stable inhibitor of cAMP-dependent protein kinase (PKI alpha) cont ains both a nuclear export signal (NES) and a high affinity inhibitory regi on that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectros copy was performed on uniformly [N-15]-labeled PKI alpha to examine its str ucture free in solution. Seventy out of 75 residues were identified, and ex amination of the C alpha H chemical shifts revealed two regions of upfield chemical shifts characteristic of alpha-helices. When PKI alpha was fragmen ted into two functionally distinct peptides for study at higher concentrati ons, no significant alterations in chemical shifts or secondary structure w ere observed. The first ordered region, identified in PKI alpha (1-25), con tains an alpha-helix from residues 1-13. This helix extends by one rum the helix observed in the crystal structure of a PKI alpha (5-24) peptide bound to the catalytic subunit. The second region of well-defined secondary stru cture, residues 35-47, overlaps with the nuclear export signal in the PKI a lpha (26-75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexib le turn containing Ile46. These four residues are critical for nuclear expo rt function. The remainder of the protein in solution appears relatively un structured, and this lack of structure surrounding a few essential and well -defined signaling elements may be characteristic of a growing family of sm all regulatory proteins that interact with protein kinases.