Regulation of differentiation and invasion in RMS by ASAP1

我们发现，敲低ArfGAP蛋白ASAP1及其同源物ASAP2和ASAP3可阻断曲美替尼诱导的FN-RMS细胞分化。我们假设这是由于其作为gtase激活蛋白（GAP）对小gtpase Arf1和Arf5的功能。为了验证这一假设，我们敲除了Arf1和Arf5。ASAP1作为一种GAP，与活性Arf结合，催化GTP水解为GDP，终止Arf信号传导。因此，如果GAP活性对于ASAP1介导的分化调控至关重要，那么敲低Arf1或Arf5将与敲低ASAP1产生相反的效果。然而，我们发现Arf1和Arf6的敲低对分化的阻断程度与ASAP1相似。因此，我们假设ASAP1也可能是Arf的一个效应因子。为了验证这一假设，我们将用GAP-dead的ASAP1和不能结合Arf的ASAP1突变体拯救ASAP1敲低，用不能结合ASAP1的Arf突变体拯救Arf敲低，并确定这些拯救对FN-RMS细胞分化的影响。我们还发现ASAP1、ASAP2和ASAP3敲低通过抑制肌原性转录因子myogenin或MEF2C的表达来阻止分化。然而，ASAP1（一种膜相关蛋白）调控转录因子表达的机制尚不清楚。我们假设ASAP家族可能通过转运膜锚定蛋白来调节转录。ASAP1与整合素和受体酪氨酸激酶的转运有关。我们将通过在增殖和分化条件下对FN-RMS细胞进行亚细胞分离来验证这一假设，并通过免疫印迹确定ASAP及其整合素和RTK靶点的亚细胞定位。我们还将通过活细胞成像在ASAP1敲低和不敲低的情况下跟踪囊泡运输。关于ASAP介导的转录调节的另一种假设是ASAP对肌动球蛋白细胞骨架的调节改变了MAP激酶信号传导。为了验证这一假设，我们将用在Src或非肌肉肌球蛋白IIA结合域突变的ASAP1来挽救ASAP1敲低，并检查对分化、MAPK信号传导和肌源性转录因子表达的影响。

We discovered that knockdown of the ArfGAP ASAP1 and its homologues ASAP2 and ASAP3 block trametinib-induced differentiation of FN-RMS cells. We hypothesized that this was due to its function as a GTPase-Activating Protein (GAP) toward the small GTPases Arf1 and Arf5. To test this hypothesis, we knocked down Arf1 and Arf5. As a GAP, ASAP1 binds to active Arf, catalyzes the hydrolysis of GTP to GDP, and terminates Arf signaling. Therefore, if GAP activity is essential for ASAP1-meditated regulation of differentiation, knockdown of Arf1 or Arf5 would have the opposite effect of knockdown of ASAP1. However, we discovered that knockdown of Arf1 and Arf6 block differentiation to a similar degree as ASAP1. Therefore, we hypothesize that ASAP1 may also be an effector of Arf. To test this hypothesis, we will rescue ASAP1 knockdown with GAP-dead ASAP1 and ASAP1 mutants that are not able to bind Arf, and rescue Arf knockdown with Arf mutants that are not able to bind ASAP1 and determine the effect of these rescues on differentiation in FN-RMS cells. We also discovered that ASAP1, ASAP2 and ASAP3 knockdowns block differentiation by suppressing expression of the myogenic transcription factors myogenin or MEF2C. However, the mechanism by which ASAP1, a membrane-associated protein, is regulating transcription factor expression is unknown. We hypothesize that the ASAP family may regulate transcription through trafficking of membrane-anchored proteins. ASAP1 has been associated with trafficking of both integrins and receptor tyrosine kinases. We will test this hypothesis by subcellular fractionation of FN-RMS cells in proliferative and differentiation conditions and determine the subcellular localization of ASAP and its integrin and RTK targets by immunoblot. We will also track vesicle trafficking by live cell imaging in the presence and absence of ASAP1 knockdown. An alternative hypothesis for ASAP-mediated regulation of transcription is that ASAP's regulation of the actomyosin cytoskeleton alters MAP kinase signaling. To test this hypothesis, we will rescue ASAP1 knockdown with ASAP1 mutated at the Src or non-muscle myosin IIA binding domains and examine the effect on differentiation, MAPK signaling, and myogenic transcription factor expression.