Jcb_200810183 629..640

The molecular chaperone Hsp90 (heat shock protein 90) maintains the conformation, stability, and activity of several key oncogenic proteins, such as EGF receptor (EGFR), cyclin-dependent kinases, Raf, and p53, which are collectively known as Hsp90 client proteins (Whitesell and Lindquist, 2005). The chaperone function of Hsp90 requires the formation of a multichaperone complex, which is dependent on the hydrolysis of ATP and ADP/ATP exchange. A geldanamycin (GA) analogue, which inhibits Hsp90 by blocking its intrinsic ATPase activity, was the first in class inhibitor of Hsp90 in human clinical trials for cancer therapy. Inhibition of Hsp90 function by GA disrupts the interaction of Hsp90 with its client proteins and leads to their proteasome pathway-dependent degradation (Zhang and Burrows, 2004). Autophagy mediates the bulk degradation of intracellular components such as macromolecule complexes and subcellular organelles through lysosomal-dependent mechanisms and is essential for the maintenance of cellular homeostasis (Meijer, 2003). Three modes of autophagy have been identified in mammals (Levine and Klionsky, 2004). Macroautophagy cargo is sequestered into a specialized double-membrane structure called an autophagosome. Autophagosomes then fuse with lysosomes, and its content is subsequently degraded by hydrolytic enzymes. Microautophagy cargo is generated by direct budding of the lysosomal membrane. In chaperone-mediated autophagy (CMA), substrates are recognized by a chaperone complex containing Hsc70 (heat shock cognate of 70 kD) and delivered into lysosomes through interactions with LAMP-2A (lysosome-associated membrane protein 2A). CMA is activated during prolonged nutrient deprivation, which results in the degradation of cytosolic proteins in a molecule by molecule fashion (Dice, 2007). Promoting the degradation of Hsp90 client proteins by inhibiting Hsp90, an important protein chaperone, has been shown to be a promising new anticancer strategy. In this study, we show that an oxazoline analogue of apratoxin A (oz-apraA), a cyclodepsipeptide isolated from a marine cyanobacterium, promotes the degradation of Hsp90 clients through chaperone-mediated autophagy (CMA). We identify a KFERQ-like motif as a conserved pentapeptide sequence in the kinase domain of epidermal growth factor receptor (EGFR) necessary for recognition as a CMA substrate. Mutation of this motif prevents EGFR degradation by CMA and promotes the degradation of EGFR through the proteasomal pathway in oz-apraA–treated cells. Oz-apraA binds to Hsc70/Hsp70. We propose that apratoxin A inhibits Hsp90 function by stabilizing the interaction of Hsp90 client proteins with Hsc70/Hsp70 and thus prevents their interactions with Hsp90. Our study provides the first examples for the ability of CMA to mediate degradation of membrane receptors and cross talks of CMA and proteasomal degradation mechanisms. Cyclodepsipeptide toxin promotes the degradation of Hsp90 client proteins through chaperone-mediated autophagy