The heme-regulated eukaryotic initiation factor 2alpha (eIF-2alpha) kinase (HRI) interacts with hsp90 in situ in rabbit reticulocyte lysate (RRL). In this report, we have examined the role of hsp90 in the maturation of newly synthesized HRI in both hemin-supplemented and heme-deficient RRL. Analysis of translating polyribosomes indicated that hsp90 interacts with nascent HRI cotranslationally. Coimmunoadsorption of HRI with hsp90 by the 8D3 anti-hsp90 antibody indicated that this interaction persisted after release of newly synthesized HRI from ribosomes. Incubation of HRI in heme-deficient lysate resulted in the transformation of a portion of the HRI polypeptides into an active heme-regulatable eIF-2alpha kinase that exhibited slower electrophoretic mobility. Transformation of HRI was dependent on autophosphorylation, and transformed HRI was resistant to aggregation induced by treatment of RRL with N-ethylmaleimide. Transformed HRI did not coimmunoadsorb with hsp90, and regulation of the activity of transformed HRI by hemin was not hsp90-dependent. The hsp90 binding drug geldanamycin disrupted the interaction of hsp90 with HRI and inhibited the maturation of HRI into a form that was competent to undergo autophosphorylation. Additionally geldanamycin inhibited the transformation of HRI into a stable heme-regulatable kinase. These results indicate that hsp90 plays an obligatory role in HRI acquiring and maintaining a conformation that is competent to become transformed into an aggregation-resistant activable kinase.

Recent studies indicate that p50cdc37 facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50 cdc37 is required for the biogenesis of the heme-regulated eIF2␣ kinase (HRI) in reticulocyte lysate. p50 cdc37 interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50 cdc37 stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50 cdc37 function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50 cdc37 interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50 cdc37 interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50 cdc37 to bind HRI and promote its activation, but did not disrupt the native association of p50 cdc37 with Hsp90. A C-terminal truncated mutant of p50 cdc37 inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50 cdc37 were detected in cultured K562 erythroleukemia cells. These results suggest that p50 cdc37 provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.The heme-regulated inhibitor (HRI) 1 of protein synthesis is a protein-serine kinase which coordinates the synthesis of globin chains with the availability of heme in reticulocytes (reviewed in Refs. 1 and 2). Under heme-deficient conditions, HRI phosphorylates the ␣-subunit of eukaryotic translational initiation factor eIF2. Phosphorylation of eIF2␣ causes an inhibition of polypeptide chain initiation and the arrest of protein synthesis, preventing the synthesis of apo-globin chains in the absence of heme. HRI is also activated under heme-replete conditions in response to a host of other adverse environmental stimuli, such as heat shock, agents that generate oxidative stress, and the presence of denatured proteins (1, 2).The biogenesis and activation of HRI into an active hemeregulatable eIF2␣ kinase requires its functional interaction with the chaperone machinery containing the 90-kDa heat shock protein (Hsp90) and the 70-kDa heat shock cognate protein (Hsc70) (3, 4). During HRI biogenesis and its subsequent transformation and activation, several discrete HRI intermediates are generated; these intermediates can be distinguished on the basis of their competence to become an active kinase in response to heme deficiency or upon treatment with sulfhydryl reactive compounds such as N-ethylmaleimide. Immediately after their synthesis, HRI molecules are not active in hemereplete or heme-deficient rabbit reticulocyte lysate (RRL) and cannot be activated by N-ethylmaleimide treatment. This immature population interacts with Hsp90 and Hsc70 (3-7). Subsequent to this immature phase, a "mature-competent" HRI population appear...

SUMMARY Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the co-chaperone p50Cdc37, Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50Cdc37 and recruitment of the ATPase activating co-chaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50Cdc37 and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50Cdc37 phosphorylation on Y4 and Y298 disrupts client-p50Cdc37 association, while Hsp90 phosphorylation on Y197 dissociates p50Cdc37 from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1 which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining co-chaperones.

Hsp90 and p50(cdc37) provide a poorly understood biochemical function essential to certain protein kinases, and recent models describe p50(cdc37) as an exclusive hsp90 cohort which links hsp90 machinery to client kinases. We describe here the recovery of p50(cdc37) in immunoadsorptions directed against the hsp90 cohorts FKBP52, cyp40, p60HOP, hsp70, and p23. Additionally, monoclonal antibodies against FKBP52 coadsorb maturation intermediates of the hsp90-dependent kinases p56(lck) and HRI, and the presence of these maturation intermediates significantly increases the representation of p50(cdc37) and hsp90 on FKPB52 machinery. Although the native heterocomplex between hsp90 and p50(cdc37) is salt-labile, their dynamic interactions with kinase substrates produce kinase-chaperone heterocomplexes which are highly salt-resistant. The hsp90 inhibitor geldanamycin does not directly disrupt the native association of hsp90 with p50(cdc37) per se, but does result in the formation of salt-labile hsp90-kinase heterocomplexes which lack the p50(cdc37) cohort. We conclude that p50(cdc37) does not simply serve as a passive structural bridge between hsp90 and its kinase substrates; instead, p50(cdc37) is a nonexclusive hsp90 cohort which responds to hsp90's nucleotide-regulated conformational switching during the generation of high-affinity interactions within the hsp90-kinase-p50(cdc37) heterocomplex.

between 17 and 64 N m -1 , using scan rates of 1-2 lines s -1 and a resolution of 512 pixels × 512 pixels. The SFM images in Figure S2 were taken with an Autoprobe CP research microscope (Thermomicroscope, Veeco, Santa Barbara, California, USA) operating at room temperature in air. Images with scan lengths ranging from 5 lm down to 0.5 lm have been recorded with a resolution of 512 pixels × 512 pixels using the 5 lm scanner and non-contact Si ultralevers with a spring constant (k) in the range 2. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] The unique properties of single-walled carbon nanotubes (SWNTs) can lead to a new generation of composites with high strength, electrical conductivity, and chemical and structural stability.[1-10] These properties are being utilized in the fabrication of many proof-of-concept applications including nanoelectronic devices, [11][12][13] sensors, [14][15][16][17][18][19] field-emission electron sources, [20,21] displays and lighting elements, [22,23] and scanning probe tips. [24] Recent reports on the functionalization of nanotubes with bioactive groups such as peptides, [25][26][27][28] proteins, [29][30][31] and DNA [32,33] have also attracted attention to the potential importance of SWNTs in biology and medicine. So far, SWNTs have mainly been utilized as sensing materials for various biological molecules such as DNA, [34] glucose, [35][36][37] peptides, and proteins. [38][39][40][41] They have also been used as molecular tips functioning as biological probes.[