Modular Folding and Evidence for Phosphorylation-induced Stabilization of an hsp90-dependent Kinase

Steven D Hartson,Elizabeth A Ottinger,Wenjun Huang,George Barany,Paul Burn,Robert L Matts

doi:10.1074/jbc.273.14.8475

Steven D Hartson, Elizabeth A Ottinger + Show 4 more

Open Access

https://doi.org/10.1074/jbc.273.14.8475

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Abstract

The de novo folding of the individual domains of the src family kinase p56(lck) was examined within the context of full-length p56(lck) molecules produced in rabbit reticulocyte lysate containing active chaperone machinery. The catalytic domain required geldanamycin-inhibitable heat shock protein 90 (hsp90) function to achieve its active protease-resistant conformation, but the src homology 2 (SH2) domain acquired phosphopeptide-binding competence independently of hsp90 function. The SH2 domain of hsp90-bound p56(lck) was folded and functional. In addition to the facilitation by hsp90 of kinase biogenesis, a conditional role in maintenance folding could be demonstrated; although wild type p56(lck) molecules with a negative-regulatory C-terminal tyrosine matured to a nearly hsp90-independent state, p56(lck) molecules with a mutated C-terminal tyrosine continued to require hsp90-mediated maintenance. De novo folding could be distinguished from maintenance folding on the basis of proteolytic fingerprints and the effects of different temperatures on folding behavior. Results indicate that during p56(lck) biogenesis, the SH2 domain rapidly folds independently of hsp90 function, followed by the slower hsp90-dependent folding of the catalytic domain and suggest the final stabilization of p56(lck) structure by phosphorylation-mediated interdomain interactions.

Highlights

Protein domains are discrete units of compact globular structure
Because hsp90 function can be inhibited by the benzoquinone anasamycin geldanamycin [16], hsp90-dependent protein folding pathways can be dissected in unfractionated rabbit reticulocyte lysates (RRL), containing active chaperone machinery [10, 15, 17,18,19]
Like monomeric [35S]p56lck, [35S]p56lck occurring in the p56lck1⁄7hsp90 complex was bound by phosphorylated mTa peptide resins in a specific fashion (Fig. 1A). This binding was qualitatively and quantitatively equivalent to that observed for the monomer produced. These results indicated that the src homology 2 (SH2) domain of hsp90-bound p56lck was folded and functional, or was capable of becoming so during binding assays

Summary

Introduction

Protein domains are discrete units of compact globular structure. They frequently retain structure and function when separated from the whole protein by proteolysis or by protein engineering and are often defined as independently folding units. The folding of the p56lck SH2 domain was independent of hsp90 function (Fig. 2), we have previously observed that geldanamycin-mediated inhibition of hsp90 function results in the production of p56lck molecules that are deficient in kinase activity and that have compromised integrity in subsequent protease nicking assays [10].

Results

Conclusion