Inhibition of Autophagy and Multiple Steps in Asialoglycoprotein Endocytosis by Inhibitors of Tyrosine Protein Kinases (Tyrphostins) (*)
- From the (1) Department of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway
- ↵§ To whom correspondence should be addressed: Dept. of Tissue Culture, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway. Tel.: 47-22-93-59-47; Fax: 47-22-73-29-44.
Abstract
In isolated rat hepatocytes, several tyrosine protein kinase inhibitors (tyrphostins) reduced the autophagic sequestration
of electroinjected [3H]raffinose by 40-75% at doses that did not significantly affect cellular ATP levels or plasma membrane integrity. Tyrphostin
46 specifically inhibited autophagy, whereas tyrphostins 1, 25 and 51 also suppressed the receptor-mediated endocytic uptake
of 
I-tyramine-cellobiose-asialoorosomucoid, 
I-TC-AOM, by 20-30% and its degradation by 70-90%. Tyrphostins 1 and 51, and the microtubule inhibitor vinblastine, inhibited
an early endocytic step (endosome maturation/multivesiculation?), causing accumulation of endocytosed 
I-TC-AOM in a recycling compartment that corresponded to light endosomes (1.10-1.11 g/ml) in sucrose density gradients. In
the electron microscope, these endosomes could be recognized as small, peripheral endocytic vesicles and tubules accumulating
endocytosed AOM-gold. The serine/threonine protein phosphatase inhibitor okadaic acid inhibited an intermediate endocytic
step (detachment of multivesicular endosomes from the tubulovesicular network?), causing accumulation of 
I-TC-AOM in a recycling compartment corresponding to light endosomes (1.10-1.11 g/ml), but with a multivesicular rather than
a tubulovesicular morphology. Tyrphostin 25 inhibited endocytosis at a late step (endosome-lysosome fusion?), causing accumulation
of 
I-TC-AOM in a non-recycling compartment corresponding to dense, multivesicular endosomes (1.14 g/ml) that had probably detached
from the light endosomal network.
Footnotes
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↵* This work was generously supported by the Norwegian Cancer Society and by the Research Council of Norway. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
