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- Materials and methods
Poly C binding protein 1 (PCBP1) is an expressional regulator of the mu-opioid receptor (MOR) gene. We hypothesized the existence of a PCBP1 co-regulator modifying human MOR gene expression by protein–protein interaction with PCBP1. A human brain cDNA library was screened using the two-hybrid system with PCBP1 as the bait. Receptor for activated protein kinase C (RACK1) protein, containing seven WD domains, was identified. PCBP1-RACK1 interaction was confirmed via in vivo validation using the two-hybrid system, and by co-immunoprecipitation with anti-PCBP1 antibody and human neuronal NMB cell lysate, endogenously expressing PCBP1 and RACK1. Further co-immunoprecipitation suggested that RACK1-PCBP1 interaction occurred in cytosol alone. Single and serial WD domain deletion analyses demonstrated that WD7 of RACK1 is the key domain interacting with PCBP1. RACK1 over-expression resulted in a dose-dependent decrease of MOR promoter activity using p357 plasmid containing human MOR promoter and luciferase reporter gene. Knock-down analysis showed that RACK1 siRNA decreased the endogenous RACK1 mRNA level in NMB, and elevated MOR mRNA level as indicated by RT-PCR. Likewise, a decrease of RACK1 resulted in an increase of MOR proteins, verified by 3H-diprenorphine binding assay. Collectively, this study reports a novel role of RACK1, physically interacting with PCBP1 and participating in the regulation of human MOR gene expression in neuronal NMB cells.
PCBP1, the mu-opioid receptor (MOR) gene regulator, belongs to the K homology (KH) domain superfamily, which is consisting of two subsets in mammalian cells: hnRNPs K/J and the αCP proteins (Makeyev and Liebhaber 2002). The αCP proteins are also known as PCBPs, including PCBP1, 2, 3, and 4, with additional isoforms generated via alternative splicing. Within the KH domain superfamily, hnRNPK is considered the prototype and well characterized. PCBP1 and PCBP2 are the major forms of PCBPs expressing in mammalian cells, while the expressions of PCBP3 and PCBP4 are comparatively less (Perera et al. 2007).
PCBP proteins are known to be involved in various biological processes, including but not limited to, mRNA stabilization, transcriptional and translational regulation (Kiledjian et al. 1997; Thakur et al. 2003; Malik et al. 2006; Rivera-Gines et al. 2006). For example, PCBP2 has been shown to regulate viral genome replication and translation (Walter et al. 2002; Zhang et al. 2007). PCBP3 can function as a repressor (Kang et al. 2012). PCBP4, also known as MCG10, can suppress cell proliferation by inducing apoptosis and cell cycle arrest (Zhu and Chen 2000). In addition, studies have shown that PCBP1 participates in regulations of the androgen receptor gene (Cloke et al. 2010), eIF4E gene (Meng et al. 2007), and the MOR gene (Ko and Loh 2005). The relationship between PCBP1 and MOR gene was originally discovered in a yeast one-hybrid study, in which PCBP1 was identified as a mouse MOR gene regulator by screening a mouse brain cDNA library with the mouse MOR single-stranded (ss) cis-element as the target (Ko and Loh 2005). Naturally, various cis-elements and factors are also involved in the MOR gene regulation (Choe et al. 1998; Ko et al. 1998, 2003; Ko and Loh 2001; Choi et al. 2005; Kim et al. 2005). The PCBP1 not only participates in mouse MOR gene regulation but also involves in human MOR gene in a positive manner, reflecting a high homology of the MOR ssDNA element between mouse and human, and the PCBP1 protein is conserved among mammals (Cook et al. 2010). In addition, PCBP1 not only can act as a positive regulator but also as a negative regulator (Zhang et al. 2010). Taken together, these data suggest that PCBP1 possesses divergent roles in regulating gene expressions.
The different functional roles of a protein can be affected by its microenvironment in a context-dependent manner, which may be mediated through variable protein–protein interactions. Therefore, we tested a hypothesis of the presence of a PCBP1-interacting protein, which can impact human MOR gene regulation via its physical interaction with PCBP1. In this study, a bacteria two-hybrid screening study was carried out using PCBP1 as the bait protein. We identified one of the PCBP1-interacting proteins as RACK1, a known tryptophan-aspartic acid (WD) containing protein (McCahill et al. 2002; Tcherkasowa et al. 2002; Imai et al. 2009). RACK1-PCBP1 interaction and the interaction domain as well as the effects of RACK1, via over-expression and siRNA knock-down, on human MOR gene expression were investigated using the human neuronal NMB cell model system.
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- Materials and methods
In this study, RACK1 was identified as a PCBP1-interacting protein via screening a human cDNA library using the bacteria two-hybrid system and by the co-immunoprecipitation assay. Effect of the functional RACK1-PCBP1 interaction on the human MOR (hMOR) gene expression was also revealed using the human NMB neuronal cell model system.
PCBP1 is known to participate in the hMOR gene regulation (Cook et al. 2010); therefore, physical interaction (Figs 2, 3) between PCBP1 and RACK1 in turn can bestow RACK1 modulating the hMOR gene expression is reasonable. This RACK1 functional role is strongly supported by the observations that (i) an increase of RACK1 by over-expression decreased the hMOR gene expression as determined using the reporter gene assay (Fig. 4a) and (ii) a decrease of RACK1 by siRNA knock-down increased the hMOR expression (Figs 4d–e and 6a, b). Interestingly, the changes of RACK1 and hMOR mRNA levels were evident at 48 and 72 h after RACK1 siRNA transfection (Figs 4b–e, 5a, and 6a), but the detectable changes at the protein level required 72 h (Figs 5b and 6b), implicating that approximately a 24-h lag is required to effectively translate the information of altered mRNA amounts into the detectable change of protein level using western blot analysis, in addition to the turnover rate of MOR protein. However, this discrepancy could also reflect the differences in detection limits and/or sensitivities between western blot and RT-PCR analyses.
RACK1 was originally identified as a receptor for activated protein kinase C (Ron et al. 1995). It contains seven Trp-Asp repeats (WD domains), which can mediate protein–protein interaction (McCahill et al. 2002; Tcherkasowa et al. 2002; Imai et al. 2009). From this study, the key interaction region of RACK1-PCBP1 is mapped at the C-terminus WD7 of RACK1 protein (Fig. 3a–b), for WD7 alone can provide the interactive capability similar to that of the full-length RACK1. In addition, the WD2 or WD3 domain alone confers a minor capability, and WD6 possesses a marginal interacting ability, whereas WD1, 4 and 5 domains contain no interactive capability. These conclusions were also supported by the serial WD deletion results, for WD1 deletion (WD2-7) showed no significant change on the cell growth as compared with the full-length RACK1 (WD1-7), supporting that WD1 contributed no PCBP1-interacting ability. Deletion of WD2 and WD3 resulted in a partial decrease of interactive ability, corroborating that there is a degree of interaction ability attributing from WD2 and WD3 domains. Interestingly, the construct containing WD4-7 displayed a further decrease of interactive ability, and the removal of WD4 (WD5-7) improved the capability to interact. These results implicates that WD4 domain may confer a negative effect, possibly a steric hindrance, on the PCBP1-RACK1 interaction ability. Particularly, WD4 itself possesses no PCBP1 interaction capability. In addition, it is also possible that WD4 may interact with a negative regulator, which can inhibit or interfere the PCBP1-RACK1 interaction. There was no significant difference between WD5-7 and WD6-7 constructs, suggesting that WD5 domain contributes no direct effect to the PBCP1-RACK1 interaction, which is collaborated with the WD5 alone possessing no interaction ability. Although WD6 alone possesses a slight interaction capability, yet the combination of WD6 and WD7 domains resulted in a decrease of interaction ability as compared to WD 7 alone, implicating a possible conformation interaction or a degree of steric hindrance between WD6 and 7. Lastly and critically, WD7 alone is sufficient for the full interactive ability with PCBP1.
The WD motif has the bladed propeller structure (Buensuceso et al. 2001; Sondek and Siderovski 2001), and can form hydrogen bonding between the β strands of the propeller (Sklan et al. 2006). In addition, the sequences of RACK1 WD domains are well conserved in various species, such as plants (Nakashima et al. 2008), Drosophila, C. elegans (Julie et al. 2007), mammals and humans (Guillemot et al. 1989). However, on the basis of the two-hybrid results using the constructs containing each single RACK1 WD domain, our data clearly suggested that the individual WD domains of RACK1 are not functionally equivalent, though each WD domain has the bladed propeller structure. There are at least two possibilities, which contribute to the uniqueness of each WD motif: (i) Each WD domain possesses different sequences (Fig. 3c), though each domain contains the WD repeat with the bladed propeller structure. (ii) The region connecting two sequential WD domains, called the loop of RACK1 (Garcia-Higuera et al. 1998), may differ in size and sequence, contributing to the unique features of each WD motif.
The WD motif may also provide a docking platform for various protein–protein interactions (Chen et al. 2004). Therefore, via the different WD domains, RACK1 may coordinate various protein interactions. RACK1 has been reported to be able to interact with several proteins, such as protein kinase C (PKC), src kinase and insulin receptor/IGF1R. The interaction site for RACK1-PKC was mapped at WD3 and/or WD5 and 6 domains (Buensuceso et al. 2001; McCahill et al. 2002). WD6 domain mediates RACK1-src interaction (McCahill et al. 2002), and WD 1-4 domains are responsible for RACK1-insulin receptor/IGF1R interaction (Zhang et al. 2006). This study provides additional evidence that RACK1 interacts with PCBP1 via WD7 domain predominantly. Taken together, RACK1 possesses capability to interact with various proteins via different WD domains. Furthermore, proteins may also interact with RACK1 at the sides, below or at its border of the propeller (Sklan et al. 2006).
There are several reports related to the functional roles of RACK1 and its interacting proteins. For example, RACK1 has been suggested to serve as a scaffold protein for PKC isoforms, and it enables translocation and stabilization of PKC isoforms (Mochly-Rosen and Gordon 1998). The reduction of RACK 1 levels was shown to correlate with malfunctioning of PKC translocation in aging rat brain (Battaini et al. 1997). It has also been shown to impair insulin-induced kinase translocation, Xenopus oocyte maturation (Ron et al. 1995) and regulation of calcium channels in cardiomyocytes (Zhang et al. 2006). A study of RACK1-src kinase interaction showed that RACK1 over-expression can decrease src activity and cell growth rates in NIH 3T3 cells (Chang et al. 1998). Collectively, these reports and our data demonstrated that RACK1 plays various functional roles depending on the particular proteins it interacts with.
Thus, based on this study using human neuronal NMB cell system, a functional role of RACK1-PCBP1 interaction and its mechanism governing the MOR gene expression are proposed in Fig. 6c. A simplified model depicts a center surrounding the dynamic interaction/equilibrium between free forms of RACK1 and PCBP1 (on left-hand side) as well as the RACK1-PCBP1 complex (on the right-hand side of the equilibrium): (i) The free form of PCBP1 can enter the nucleus and is known to act as transcriptional regulator of MOR gene by increasing MOR transcription (Ko and Loh 2005; Malik et al. 2006; Cook et al. 2010). (ii) When an elevated amount of RACK1 is achieved via over-expression, RACK1 interacts with PCBP1, shifting the equilibrium toward the right-hand side to generate more RACK1-PCBP1 complexes, reducing the available amount of free PCBP1, and then resulting in a decrease of MOR gene expression. (iii) Conversely, when a decrease of endogenous RACK1 level is achieved via RACK1 siRNA knock-down, the equilibrium is then driven toward the left-hand side, resulting in more free form of PCBP1, increasing MOR mRNA expression (iv), which then in turn increases the MOR protein level via post-transcriptional events (v) as well as translation and post-translational events (vi).
In addition, this study also found that RACK1 was mainly distributed in the cytosol with a few hot spots in the nuclei of NMB cells (Fig. 1b, indicated by arrows). The hot spots in the nucleus may suggest a potential functional role of RACK1 in the nucleus, though its functional role in the nucleus is unknown for now. Furthermore, PCBP1 is also expressed in the entire cell with many hot spots found in the nucleus of neuronal cells (Berry et al. 2006). Although these two proteins are distributed throughout the entire cell, importantly, PCBP1-RACK1 interaction takes place in the cytosol, but not in the nucleus of NMB cells (Fig. 2c). The actual reason is unknown; however, there are several potential explanations. For example, the change of a protein's phosphorylation/dephosphorylation status may influence its protein–protein interaction ability. It has been reported that PCBP1 nuclear retention is enhanced via its phosphorylation (Meng et al. 2007). Therefore, the protein–protein interaction ability of phosphorylated PCBP1 may be compromised in the nucleus. In addition, the phosphorylated/dephosphorylated RACK1 may also possess different protein–protein interaction ability. A second possibility is protein compartmentalization, with the protein in a particular microenvironment not available for the protein–protein interaction. RACK1 demonstrates the punctate staining in the nucleus (Fig. 1b), suggesting that RACK1 may be compartmentalized and thus not available for interacting with PCBP1 in the nucleus. Still another possibility is that RACK1 may directly or indirectly regulate MOR gene expression via an unidentified factor in the nucleus. These possibilities will need to be further investigated.
In summary, this study shows that RACK1 is a PCBP1-interacting protein and can modulate hMOR gene regulation as determined using the human NMB neuronal cell model system. This new functional role of PCBP1-RACK1 interaction provides some insight into MOR regulation and may be helpful to develop potential strategies of alternating MOR gene expression.