A Proline Residue in the α-Helical Rod Domain of Type I Keratin 16 Destabilizes Keratin Heterotetramers*
- From the Departments of Biological Chemistry and Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Abstract
The type I keratins 14 (K14) and 16 (K16) are distinct in their assembly properties and their expression pattern despite a high degree of sequence identity. Understanding K16 function and regulation is of interest, given its strong induction in keratinocytes located at the wound edge after injury to stratified epithelia. We reported previously that, compared with K14, K16 forms unstable heterotetramers with either K5 or K6 as the type II keratin pairing partner (Paladini, R. D., Takahashi, K., Bravo, N. S., and Coulombe, P. A. (1996) J. Cell Biol. 132, 381–397). We show here that yet another related type I keratin, K17, forms stable heterotetramers with a variety of type II keratins, further accentuating the unique nature of K16. Analysis of chimeric K14-K16 proteins in a heterotetramer formation assay indicated that the instability determinant resides in a 220-amino acid segment within the α-helical rod domain of K16. Site-directed mutagenesis revealed that Pro188, an amino acid residue located in subdomain 1B of the rod, accounts quantitatively for the instability of K16-containing heterotetramers under denaturing conditions. In vitropolymerization studies suggest that the presence of Pro188correlates with a reduction in assembly efficiency. In addition to their implications for the stable conformation of the keratin heterotetramers, these findings suggest that the tetramer-forming properties of K16 may influence its partitioning between the soluble and polymer pools, and hence contribute to its regulation in epithelial cells under resting and wound repair conditions.
Footnotes
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↵* This work was supported in part by National Science Foundation Grant MCB-9319560 and National Institutes of Health Grant AR44232.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Both authors contributed equally to this work.
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↵§ Recipient of a Junior Faculty Research Award from the American Cancer Society. To whom correspondence should be addressed: Dept. of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-614-0510; Fax: 410-955-5759; E-mail: pacoulom{at}welchlink.welch.jhu.edu.
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↵1 The abbreviations used are: IF, intermediate filament; K, keratin; PCR, polmerase chain reaction; PAGE, polyacrylamide gel electrophoresis; β-ME, β-mercaptoethanol.
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↵2 P. Sessiah, J. Fradette, and P. A. Coulombe, unpublished data.
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↵3 K. McGowan and P. A. Coulombe, unpublished data.
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↵4 J. Fradette and P. A. Coulombe, unpublished data.
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↵5 R. D. Paladini and P. A. Coulombe, unpublished observations.
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- Received July 30, 1997.
- Revision received September 29, 1997.
