HDAC6 Regulates EGFR Trafficking Figure 1. HDAC6 was up-regulated in Pkd1 mutant renal epithelial cells and kidney tissues. (A) Western blotting analysis of the expression of HDAC6 in Pkd1 wild-type (WT) and Pkd1null/null (Null) MEK cells as well as in postnatal Pkd1 heterozygous PH2 cells and homozygous PN24 cells. The expression of HDAC6 in the above cells was quantified from three independent immunoblots and was standardized to actin. ** p , 0.01. (B) The activity of HDAC6 in Pkd1 wild-type (WT) and Pkd1null/null (Null) MEK cells as well as in postnatal Pkd1 heterozygous PH2 cells and homozygous PN24 cells. The activity of HDAC6 was increased about 7.8 fold in Pkd1null/null (Null) MEK cells and 4.8 fold in postnatal Pkd1 homozygous PN24 cells compared with that in Pkd1 wild type MEK cells and postnatal Pkd1 heterozygous PH2 cells, respectively. The HDAC6 activity assay was performed in three independent experiments. * p , 0.05, ** p , 0.01. (C) Western blotting analysis of the expression of HDAC6 in Pkd1 wild type and Pkd1 hypomorphic homozygous (Pkd1nl/nl) kidney tissues collected at postnatal days 7, 14, and 21. The relative expression levels of HDAC6 in different kidneys were standardized to actin and were presented in the lower panel. (D) Immunohistochemistry of HDAC6 in kidney sections from Pkd1 wild type and Pkd1nl/nl mice harvested at postnatal day 28. Scale bar, 20 mm. doi:10.1371/journal.pone.0049418.g001 HDAC activity accelerated the transport of the activated EGFR from the early endosomes to late endosomes/lysosomes. Microtubule Stability Affected EGF Induced EGFR Degradation Deacetylation of a-tubulin by HDAC6 has been reported to destabilize microtubules and decrease motor binding.[19,24] To determine whether microtubule stability affects EGFR degrada- tion in renal epithelial cells, we treated Pkd1 wild type and mutant MEK cells with nocodazole, which depolymerizes microtubules. Since ligand-induced degradation is the primary mechanism that controls EGFR levels, we also determined if microtubule stability affects EGFR degradation in response to EGF. Pkd1 wild type and mutant MEK cells were treated with 0.2 mg/ml of nocodazole and/or 100 ng/ml of EGF. As expected, EGF alone induced the degradation of EGFR in Pkd1 wild type and mutant MEK cells. However, EGF-induced EGFR degradation was strikingly atten- uated in Pkd1 wild type and mutant MEK cells treated with nocodazole for 2 hours (Fig. 8A and 8B). We further found that treatment with nocodazole strikingly decreased the transport of EGFR from early endosomes to late endosomes in Pkd1 wild type and mutant MEK cells (p , 0.05) (Fig. 9A-9D). These results together with the finding that HDAC6 deficiency induces microtubule hyper-acetylation and promotes microtubule-mediat- ed endocytic vesicle transport [16,20] suggest that microtubule- mediated endocytic vesicle transport affects EGF induced EGFR degradation. PLOS ONE | www.plosone.org 3 November 2012 | Volume 7 | Issue 11 | e49418