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

Published online by Cambridge University Press:  01 March 1999

JENNIFER A. HAUER
Affiliation:
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0654
PHILIPPE BARTHE
Affiliation:
Centre de Biochimie Structurale CNRS UMR 9955, INSERM U414, University of Montpellier I, 15 Av. Charles Flahault, 34060 Montpellier Cedex 1, France
SUSAN S. TAYLOR
Affiliation:
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0654
JOSEPH PARELLO
Affiliation:
UPRESA, 5-74 CNRS, University of Montpellier I, France, and Cancer Research Center, The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, California 92037
ANDRÉ PADILLA
Affiliation:
Centre de Biochimie Structurale CNRS UMR 9955, INSERM U414, University of Montpellier I, 15 Av. Charles Flahault, 34060 Montpellier Cedex 1, France
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Abstract

The heat stable inhibitor of cAMP-dependent protein kinase (PKIα) contains both a nuclear export signal (NES) and a high affinity inhibitory region that is essential for inhibition of the catalytic subunit of the kinase. These functions are sequentially independent. Two-dimensional NMR spectroscopy was performed on uniformly [15N]-labeled PKIα to examine its structure free in solution. Seventy out of 75 residues were identified, and examination of the CαH chemical shifts revealed two regions of upfield chemical shifts characteristic of α-helices. When PKIα was fragmented into two functionally distinct peptides for study at higher concentrations, no significant alterations in chemical shifts or secondary structure were observed. The first ordered region, identified in PKIα (1–25), contains an α-helix from residues 1–13. This helix extends by one turn the helix observed in the crystal structure of a PKIα (5–24) peptide bound to the catalytic subunit. The second region of well-defined secondary structure, residues 35–47, overlaps with the nuclear export signal in the PKIα (26–75) fragment. This secondary structure consists of a helix with a hydrophobic face comprised of Leu37, Leu41, and Leu44, followed by a flexible turn containing Ile46. These four residues are critical for nuclear export function. The remainder of the protein in solution appears relatively unstructured, and this lack of structure surrounding a few essential and well-defined signaling elements may be characteristic of a growing family of small regulatory proteins that interact with protein kinases.

Type
Research Article
Copyright
© 1999 The Protein Society

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