Document Type

Dissertation - Open Access

Award Date

2017

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Adam Hoppe

Abstract

Macrophage colony stimulating factor receptor (CSF-1R or MCSFR) is a receptor tyrosine kinase essential for the growth and function of macrophages. Understanding the mechanisms that regulate CSF-1R activation and deactivation will provide insights to clinical treatment of macrophage related diseases including chronic inflammation and cancer. Previously, our laboratory showed that CSF-1R undergoes a novel membrane trafficking route that involves macropinocytosis to deactivate CSF-1R signaling. This thesis makes the discovery that the ubiquitin ligases Cbl and Cbl-b cooperate to regulate CSF-1R endocytosis and traffic to macropinosome in macrophages. Macrophages were derived from mice knocked out for Cbl, Cbl-b or the double knock out (DKO). DKO macrophages hyperproliferated, matching the severe myeloproliferative disorder observed in DKO mice. The CSF-1R and associated proteins were not ubiquitinated in DKO macrophages, unlike single knockouts and wild-type cells suggesting redundant functions of Cbl and Cbl-b. Mapping of the CSF-1R traffic demonstrated that CSF-1R internalization was slower in DKO cells, resulting in prolonged CSF-1R signaling at the plasma membrane and prolonged Akt signaling. Interestingly, CSF-1R transport to the lumen of macropinosome was defective in DKO cells, suggesting that altered membrane transport is responsible for the cellular phenotype. Tyrosine phosphorylation was drastically decreased and ERK signaling was lower in DKO macrophages, possibly x resulting from defective CSF-1R signaling on endosome and macropinosome. One of the key ESCRT proteins, HRS, did not associate with the CSF-1R in DKO macrophages indicating Cbl and Cbl-b are required for ESCRT-mediated transport into the lumen of macropinosome. Surprisingly, the CSF-1R was still degraded in DKO cells by an unknown mechanism. RNA sequencing analysis showed that Cbl and Cbl-b work together to regulate approximately 1,300 genes, while Cbl appears to regulate a unique set of approximately 250 genes in macrophages. In conclusion, Cbl and Cbl-b share partially redundant functions regulating CSF-1R signaling, endocytic traffic and cell growth.

Library of Congress Subject Headings

Colony-stimulating factors (Physiology)
Macrophages.
Endocytosis.
Ligases.

Description

Includes bibliographical references (pages 72-87)

Format

application/pdf

Number of Pages

97

Publisher

South Dakota State University

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Rights Statement

In Copyright