Evolutionarily conserved dual lysine motif determines the non-chaperone function of secreted Hsp90alpha in tumour progression.

Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (α and ß) have been shown to support tumour progression. The tumour-supporting activity of the intracellular Hsp90 is attributed to their N-terminal ATPase-driven chaperone function. What molecular entity determines the extracellular function of secreted Hsp90 and the distinction between Hsp90α and Hsp90ß was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90α nullifies tumour cells' ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90ß leads to tumour cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90ß, protein recovers tumourigenicity of the Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90ß subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90ß with lysines convert Hsp90ß to a Hsp90α-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α inhibits both de novo tumour formation and expansion of already formed tumours in mice. This study suggests an alternative therapeutic approach to target Hsp90 in cancer, that is, the tumour-secreted Hsp90α, instead of the intracellular Hsp90α and Hsp90ß.

NMR analysis of structure and dynamics of the cytosolic tails of integrin alpha IIb beta 3 in aqueous solution.

The structural and dynamic properties of the cytosolic tails of the adhesion receptor integrin alphaIIbbeta3, fused to a coiled-coil construct via (Gly)(3) linkers, were studied in aqueous solution by nuclear magnetic resonance (NMR) spectroscopy. Both tails were largely flexible and unstructured, although, in the beta3 tail, residues Arg(724)-Ala(735) have a propensity to form a helical structure and residues Asn(744)-Tyr(747) (NPLY) have a propensity to adopt reverse-turn conformations. The mutation beta3(Y747A) disrupted this reverse-turn tendency and markedly reduced the affinity of the head domain of the cytoskeletal protein, talin for the beta3 tail. Omission of the (Gly)(3) linker connecting the coiled-coiled helices and the integrin tails lead to helix propagation into the beta3 tail extending up to eight residues. A variety of different tail constructs were made and studied to reveal tail-tail interactions, but surprisingly no significant interactions between both tails could be detected within the context of our constructs. These results provide structural insight into a highly conserved beta tail motif (NPXY/F) required for integrin signaling and highlight a second transiently structured region (residues Arg(724)-Ala(735)), which might also be of functional significance.

A membrane-distal segment of the integrin alpha IIb cytoplasmic domain regulates integrin activation.

Previous evidence suggests that interactions between integrin cytoplasmic domains regulate integrin activation. We have constructed and validated recombinant structural mimics of the heterodimeric alpha(IIb)beta(3) cytoplasmic domain. The mimics elicited polyclonal antibodies that recognize a combinatorial epitope(s) formed in mixtures of the alpha(IIb) and beta(3) cytoplasmic domains but not present in either isolated tail. This epitope(s) is present within intact alpha(IIb)beta(3), indicating that interaction between the tails can occur in the native integrin. Furthermore, the combinatorial epitope(s) is also formed by introducing the activation-blocking beta(3)(Y747A) mutation into the beta(3) tail. A membrane-distal heptapeptide sequence in the alpha(IIb) tail ((997)RPPLEED) is responsible for this effect on beta(3). Membrane-permeant palmitoylated peptides, containing this alpha(IIb) sequence, specifically blocked alpha(IIb)beta(3) activation in platelets. Thus, this region of the alpha(IIb) tail causes the beta(3) tail to resemble that of beta(3)(Y747A) and suppresses activation of the integrin.

The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases.

The regulatory fragment of Src kinases, comprising Src homology (SH) 3 and SH2 domains, is responsible for controlled repression of kinase activity. We have used a multidisciplinary approach involving crystallography, NMR, and isothermal titration calorimetry to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase that contains both phosphotyrosine and polyproline motifs. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and focal adhesion kinase is restricted to the canonical SH3 and SH2 binding sites and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site while maintaining rapid dissociation after signaling.