Session Time: 3:15pm-4:45pm
Presentation Time: 3:39pm-3:51pm
*Purpose: In our previous studies, we identified hematopoietic cell kinase (HCK) as a key driver of renal fibrosis through TGFβ/Smad3 pathway. Here in this study, we are studying the mechanism of how HCK regulates TGFβ/Smad3 pathway.
*Methods: We performed mass spectrometry (LC/MS) on 293T cell lysates immunoprecipitated with V5-taged overexpression of either HCK, or FYN or SRC. Western blot was then performed to verify the proteins binding and phosphorylation by HCK. Autophagy LC3 hiBiT reporter assay was used with HCK over-expression to measure autophagy level.
*Results: We identified 339, 403 and 715 proteins binding to FYN, HCK and SRC, respectively. The top ranked 6 proteins are listed in Table 1. Interestingly, we identified ATG2A and CBL as the top-ranked proteins for HCK but not for SRC and FYN. ATG2A and CBL are critical proteins for autophagosome formation. We confirmed the interaction of HCK with CBL, ATG2A by immunoprecipitation and western blot (Figure 1). Next, we measured the autophagy flux by a LC3 HiBiT reporter assay system from Promega (Figure 2A). We found that over-expression of HCK decreased autophagy flux while knock-down of HCK increased it (Figure 2B). While over-expression of SRC also leaded to mild increase of autophagy, over-expression of FYN didn’t have any effect on autophagy. Then, we over-expressed HCK and used IP to pull down HCK and binding proteins to detect phosphorylated proteins. We found CBL get phosphorylated with HCK over-expression(Figure 2C). As autophagy is known to attenuates tubulointerstital fibrosis through regulating transforming growth factor-β pathway, we identified a new mechanism that activated HCK promote kidney fibrosis through TGFβ/Smad3 pathway by inhibiting autophagy pathway.
*Conclusions: HCK promotes kidney fibrosis through autophagy pathway.
To cite this abstract in AMA style:Wei C, Mou L, Banu K, Menon M, Zhang W, He C, Murphy B. Hematopoietic Cell Kinase Promotes Kidney Fibrosis Through Autophagy Pathway [abstract]. Am J Transplant. 2020; 20 (suppl 3). https://atcmeetingabstracts.com/abstract/hematopoietic-cell-kinase-promotes-kidney-fibrosis-through-autophagy-pathway/. Accessed June 23, 2021.
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