A dual role of EGFR protein tyrosine kinase signaling in ubiquitination of AAV2 capsids and viral second-strand DNA synthesis.
A 52 kd cellular protein, FK506-binding protein (FKBP52), phosphorylated at tyrosine residues by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK), inhibits adeno-associated virus 2 (AAV2) second-strand DNA synthesis and transgene expression. FKBP52 is dephosphorylated at tyrosine residues by T-cell protein tyrosine phosphatase (TC-PTP), and TC-PTP over-expression leads to improved viral second-strand DNA synthesis and improved transgene expression. In these studies, we observed that perturbation of EGFR-PTK signaling by a specific inhibitor, Tyrphostin 23 (Tyr23), augmented the transduction efficiency of the single-stranded AAV (ssAAV) vector as well as the self-complementary AAV (scAAV) vector. Similarly, tyrosine-dephosphorylation of FKBP52 by TC-PTP resulted in increased transduction by both vectors. These data suggested that EGFR-PTK signaling also affects aspects of AAV transduction other than viral second-strand DNA synthesis. We document that inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsids which, in turn, facilitates nuclear transport by limiting proteasome-mediated degradation of AAV vectors. We also document that Tyr23-mediated increase in AAV2 transduction efficiency is not further enhanced by a specific proteasome inhibitor, MG132. Thus, EGFR-PTK signaling modulates ubiquitin (Ub)/proteasome pathway-mediated intracellular trafficking as well as FKBP52-mediated second-strand DNA synthesis of AAV2 vectors. This has implications in the optimal use of AAV vectors in gene therapy.
Zhong
Li
L
Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida, USA.
Zhao
Weihong
W
Wu
Jianqing
J
Li
Baozheng
B
Zolotukhin
Sergei
S
Govindasamy
Lakshmanan
L
Agbandje-McKenna
Mavis
M
Srivastava
Arun
A
D001692:Biological Transport; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D015398:Signal Transduction; D019076:Transgenes / Q000235:genetics; D054875:Ubiquitination
D036022:Capsid Proteins; D004279:DNA, Viral; D061988:Proteasome Inhibitors; D066246:ErbB Receptors; D046988:Proteasome Endopeptidase Complex