Molecular Modeling and Deletion Mutagenesis Implicate the Nuclear Translocation Sequence in Structural Integrity of Fibroblast Growth Factor-1

doi:10.1074/jbc.271.43.26876

Molecular Modeling and Deletion Mutagenesis Implicate the Nuclear Translocation Sequence in Structural Integrity of Fibroblast Growth Factor-1*

From the ^‡ Center for Cancer Biology and Nutrition, Albert B. Alkek Institute of Biosciences and Technology, Department of Biochemistry and Biophysics, Texas A & M University, Houston, Texas 77030-3303, the
^§ Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, and the Departments of
^¶ Experimental Pathology and
^∥ Molecular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855

^″; To whom correspondence should be addressed:
Center for Cancer Biology and Nutrition, Albert B. Alkek Institute of Biosciences and Technology, Dept. of Biochemistry and Biophysics, Texas A & M University, 2121 W. Holcombe Blvd., Houston, TX 77030-3303.
Tel.: 713-677-7522; Fax: 713-677-7512; E-mail: wmckeeha{at}ibt.tamu.edu

Abstract

The sequence NYKKPKL in the NH₂ terminus of fibroblast growth factor (FGF)-1 has been proposed to affect the long term activities of FGF-1 through its function as a nuclear translocation signal or its role in stabilization of the structure required to sustain binding and activation of the transmembrane receptor kinase. A dynamic molecular model of FGF-1 docked into a duplex of the FGF receptor ectodomain and a hexadecameric heparin chain suggests that the NYKKPKL sequence does not directly interact with heparin or the receptor, but rather the lysine-leucine residues within the sequence indirectly stabilize a major receptor-binding domain. Concurrent with a marked increase in dependence on exogenous heparin for optimal activity, sequential deletion of residues in the NYKKPKL sequence in FGF-1 resulted in a progressive loss of thermal stability, resistance to protease, mitogenic activity, and affinity for the transmembrane receptor. The largest change resulted from deletion of the entire sequence through the lysine-leucine residues. In the presence of sufficiently high concentrations of heparin, the deletion mutants exhibited mitogenic activity equal to wild-type FGF-1. The results confirm that a primary role of the NYKKPKL sequence domain is to maintain the structural integrity of FGF-1 required for optimal binding to and activation of the heparan sulfate-transmembrane receptor complex.

Footnotes

↵* This work was supported by NIDDK, National Institutes of Health (NIH), Grants DK35310 and DK38639 and NCI (NIH) Grant CA59971 (to W. L. M.); NHLBI (NIH) Grant HL52753 (to X. Z.); and NHLBI (NIH) Grants HL32348 and HL35627 (to T. M.) 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.
↵¹ The abbreviations used are:

FGF

fibroblast growth factor

PBS

phosphate-buffered saline

MK

mouse keratinocyte(s).
↵² M. Kan, F. Wang, M. Kan, J. L. Gabriel, and W. L. McKeehan, submitted for publication.
↵³ Y. Luo, W. Lu, F. Wang, M. Kan, J. L. Gabriel, and W. L. McKeehan, manuscript in preparation.
- Received April 5, 1996.
- Revision received July 15, 1996.