Association of Hsp90 with Cellular Src-Family Kinases in a Cell-Free System Correlates with Altered Kinase Structure and Function

Abstract

Following synthesis in the cytoplasm, the transforming proteins encoded by the retroviral oncogenes src, yes, fps, fes, and fgr form complexes with hsp90 and the hsp90 cohort p50. These cytoplasmic complexes are intermediates in the production of the mature membrane-associated kinase. However, soluble complexes between the nascent cellular homologs of these proteins and hsp90-p50 have not been readily detected [Brugge, J.S. (1986) Curr. Top. Microbiol. Immunol. 123, 1-22 and references therein]. In this paper, we have utilized protein synthesis in reticulocyte lysate to determine whether three cellular members of the src family of tyrosine kinases, myeloid-specific p59fgr, B cell-specific p59fgr, and p56lck, form complexes with hsp90. Following their synthesis, fast- and slow-sedimenting forms of these proteins can be separated on glycerol gradients. Anti-hsp90 monoclonal antibodies co-immunoadsorb the fast-sedimenting, but not the slow-sedimenting, forms of these kinases from gradient fractions. These hsp90 complexes can be detected in the complete absence of detergent. Conversely, an unrelated protein, firefly luciferase, does not form stable complexes with hsp90 following synthesis in reticulocyte lysate. Anti-p56lck antibodies specifically co-immunoadsorb hsp90 from protein synthesis reactions programmed with lckRNA. The fast-sedimenting, complex-bound form of p56lck is deficient in autophosphorylation activity and phosphorylates an exogenous substrate, acid-treated enolase, less efficiently than does the monomeric form. Fast-sedimenting p56lck is hypersentitive to limited proteolysis by chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Hsp90 Is Essential for the Synthesis and Subsequent Membrane Association, But Not the Maintenance, of the Src-Kinase p56lck

“…This could be explained by a transient interaction of Hsp90 with Lck during synthesis, in which case only a small amount of the total Lck is Hsp90 associated at steady state. Interaction with Hsp90 was demonstrated previously for Lck in reticulocyte lysates (Hartson and Matts, 1994) and in LSTRA cells (Hartson et al, 1996), a murine T-cell line that overexpresses Lck 40-fold. In both situations, the large amount of newly synthesized Lck might facilitate the detection of Hsp90 interaction.…”

“…However, these interactions could not be detected for c-Src in cells (Iba et al, 1984;Brugge, 1986), and it remained unclear whether Hsp90 was required for the synthesis of Src-kinases in general or of virally encoded Srckinases exclusively. The role of Hsp90 was further studied in rabbit reticulocyte lysates, in which the Src-kinases Fgr and Lck were found to interact with Hsp90 during in vitro translation (Hartson and Matts, 1994). This interaction was transient, and inhibition of Hsp90 activity resulted in enhanced protease sensitivity and loss of Lck kinase activity (Hartson et al, 1996).…”

“…Hsp90 has also been found to interact with the products of several viral oncogenes including the Src-kinases v-Src (Brugge et al, 1981;Oppermann et al, 1981) and v-Fgr (Ziemiecki et al, 1986). The cellular forms of both Lck and Fgr have been reported to interact with Hsp90 during and after in vitro translation (Hartson and Matts, 1994). In addition, the catalytic activity of a constitutively active form of Lck appears to require Hsp90 when the protein is expressed in fibroblasts (Hartson et al, 1996).…”

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

“…Plasmids-The coding sequences for protein domains and subdomains were cloned by PCR into a modified pSP64T plasmid (40) as previously described (24,(41)(42)(43). N-terminally His-tagged versions of each domain were constructed.…”

Molecular characteristics of immunogenic cancer cell death

“…43 Intracellular HSP90 also exerts prominent cytoprotective functions. HSP90 can bind to numerous signaling proteins, including steroid receptors, MyoD, 44 v-Src 45 or Raf-1. 46 When bound to these proteins, HSP90 ensures the correct conformational maturation of these receptors.…”