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  • It is interesting that SEC significantly inhibited cell migr

    2022-07-01

    It is interesting that SEC significantly inhibited cell migration and EMT in PC3 cells expressing a low RKIP level and HEK293T RKIP−/− cells, while had no effect on HEK293T RKIP+/+ cells. These observations can be interpreted that the RKIP-ANXA7 association may block SEC-induced ANXA7 GTPase activation. It is well known that RKIP could reverse signaling pathways by binding to its target proteins: (a) The RKIP-Raf-1 association inhibited Raf-1 phosphorylation induced by PAK and Src, and blocked the MAPK pathway; (b) RKIP interfered NF-κB signaling by binding to IKK, TAK1 and NIK; and (c) the interaction between RKIP and GRK2 inhibited the binding of GRK with its receptor substrate and contributed to the activation of GPCR signal pathway [18,58,61]. Here, we focused on the effect of RKIP on AMPK/mTORC1/STAT3 pathway mediated by ANXA7 GTPase. Our results revealed that RKIP binds to ANXA7 in HEK293T RKIP+/+ cells, which blocks the enhanced binding of ANXA7 to AMPK with SEC treatment and results in the inhibitory effect on the downstream signaling pathway. In contrary, the activation of ANXA7 GTPase by SEC activates AMPK/mTORC1/STAT3 pathway successfully in PC3 cells expressing a low RKIP level and HEK293T RKIP−/- cells. Our findings indicate that the RKIP-ANXA7 association is the key point that inhibits SEC-induced ANXA7 GTPase activation and further blocks AMPK/mTORC1/STAT3 signaling pathway. More work is needed to determine the explicit mechanism by which RKIP inhibits the activation of ANXA7 GTPase induced by SEC.
    Disclosure of potential conflicts of interest
    Introduction “Pseudoenzymes” comprise an estimated 10%–15% of known members of most enzyme classes (Murphy et al., 2017a, Pils and Schultz, 2004). These proteins adopt the overall fold of their respective enzyme families but are sequentially divergent in conserved enzymatic residues. This often results in partial or complete deficiencies in catalytic activity (Eyers and Murphy, 2016). Nonetheless, pseudoenzymes can function in signal transduction pathways as adaptors or regulators of signaling, and the study of these proteins has led to a better understanding of the roles of enzymatic folds beyond catalysis (Murphy et al., 2017b). The best studied pseudoenzymes are the pseudokinases (Boudeau et al., 2006), which were identified by genome-wide sequence analyses that revealed kinases lacking one or more of the conserved amino Resiniferatoxin motifs responsible for catalytic activity (Manning et al., 2002). Similarly, a number of pseudoenzymes within the Ras superfamily of small GTPases have been discovered (Basilico et al., 2014, Foster et al., 1996, Schroeder et al., 2014, Soundararajan et al., 2007, Splingard et al., 2007, Stiegler and Boggon, 2017), and have been termed the pseudoGTPases. Small GTPases are key regulators of cellular signaling pathways, and are classified into five subgroups: Ras, Rho, Rab, Ran, and Arf (Wennerberg et al., 2005). These proteins undergo “GTP cycling”; they bind GTP, enzymatically hydrolyze the γ-phosphate of GTP to form GDP and inorganic phosphate, then release the bound GDP and phosphate to allow binding of a new GTP molecule (Vetter and Wittinghofer, 2001). Thus, GTPases act as molecular switches with ON (GTP-bound) and OFF (GDP-bound) states. GTP cycling is intrinsically slow, so facilitator proteins help accelerate specific steps of the process; hydrolysis by GTPase activating proteins (GAPs), and GDP dissociation by guanine nucleotide exchange factors (GEFs). This cycling process occurs in each of the five GTPase subgroups, and specific amino acid residues that are important for nucleotide binding and catalysis are conserved among the superfamily. These conserved residues are termed G motifs (G1 to G5) (Bourne et al., 1991, Dever et al., 1987). By definition, pseudoGTPases lack, or are mutated, in one or more of the consensus G motifs (Murphy et al., 2017b). RhoA is the founding member of the Rho subfamily of small GTPases, and plays important roles in actin reorganization, cell migration, shape, adhesion, cytokinesis, and many other cellular functions (Jaffe and Hall, 2005). Proper regulation of RhoA signaling is therefore critical for many cellular processes. Chief among RhoA regulators are the p190RhoGAP proteins, p190RhoGAP-A (ARHGAP35) and p190RhoGAP-B (ARHGAP5) (Burbelo et al., 1995, LeClerc et al., 1991, Settleman et al., 1992). These Resiniferatoxin GAPs may account for up to 60% of RhoGAP activity in the cell (Vincent and Settleman, 1999) and are critical, for example, for proper regulation of cytoskeletal structure and contractility (Arthur and Burridge, 2001, Chang et al., 1995, Ridley et al., 1993), but, surprisingly, their molecular architecture is not well studied. Within these ∼170-kDa multidomain proteins we recently discovered two pseudoGTPase domains in a region previously thought to be flexible (Stiegler and Boggon, 2017) (Figure 1A). We also found that, although RhoGAP activity is primarily driven by the C-terminal GAP domain (Burbelo et al., 1995, LeClerc et al., 1991, Settleman et al., 1992), the newly discovered pseudoGTPase domains also impact RhoGAP activity (Stiegler and Boggon, 2017). These insights into p190RhoGAP proteins led us to consider whether there are further surprises hidden within their molecular architecture. We noted that, upon the discovery of p190RhoGAP proteins, an N-terminal GTPase fold domain had been identified (Settleman et al., 1992). However, prior investigations of this domain yielded varying results with respect to potential nucleotide binding and/or hydrolysis activities (Foster et al., 1994, Roof et al., 2000, Tatsis et al., 1998). Therefore, we surmised that a molecular level study of this domain might provide further insights into the p190RhoGAP proteins.