In this study, we investigated the effects of the anti-retroviral drug lopinavir on protein synthesis and signaling events related to the control of elongation. Our results demonstrate that the basal rate of protein synthesis declined after a relatively acute exposure of myocytes to lopinavir. The rapid effect of this drug was in contrast to previous studies, where cells required a significantly longer treatment (24-48 h) to other HIV-protease inhibitors in order to exert an inhibitory effect [Hong-Brown et al., 2004
; Hong-Brown et al., 2005
; Janneh et al., 2003
]. Our results are also in contrast to reports where levels of proteins such as P-glycoprotein immunoreactive protein were induced following extended exposure to this drug [Vishnuvardhan et al., 2003
], indicating that the synthesis of all proteins is not uniformly suppressed. Although lopinavir impaired protein synthesis, it is noteworthy that this drug did not appear affect cell viability. This is in agreement with previously reports in which treatment of kidney cells with lopinavir did not alter cell number, even after several days of drug exposure [Vidal et al., 2006
]. Taken together, our findings and published data indicate that protease inhibitors can negatively influence protein metabolism in a variety of cell types.
The mechanisms by which lopinavir alters muscle protein synthesis have not been investigated previously. In general, regulation of protein synthesis involves changes in the phosphorylation state of several key components of the translation machinery including the phosphorylation of the elongation factor eEF2. Although we did not directly determine rates of elongation in the current study, these results are consistent with the observed reduction in protein synthesis in response to lopinavir. The effect of lopinavir on eEF2 is in agreement with previous studies examining various stressors. For example, treatment with the protease inhibitor indinavir decreased the activity of eEF2 in myocytes [Hong-Brown et al., 2004
]. Likewise, alcohol or ATP depletion had a similar effect on the phosphorylation state of this elongation factor [Hong-Brown et al., 2007
; McLeod and Proud, 2002
In the present study, we provide evidence that eEF2K is an upstream regulator of eEF2. For example, lopinavir increased eEF2K phosphorylation at Ser 366 and it also increased eEF2K activity. This appears in contrast to others reports where phosphorylation at this same site was correlated with decreased eEF2K activity [Browne and Proud, 2002
; Hong-Brown et al., 2007
; Wang et al., 2001
]. Hence, it is possible that phosphorylation of other eEF2K residues, such as Ser 398, may be responsible for increased kinase activity (Browne et al., 2004
). Nevertheless, our in vivo and in vitro data showed that eEF2K activity increased in response to lopinavir, regardless of the sites that were phosphorylated.
Our studies also suggest that eEF2K is not necessarily required for the control of eEF2 phosphorylation. As such, treatment with the inhibitor rottlerin did not prevent the lopinavir-induced increase in eEF2 phosphorylation (), although it did suppress the stimulatory effect of lopinavir on eEF2K (). This idea is further supported by our in vitro kinase assay in which we used eEF2 as a substrate to directly measure lopinavir-induced changes in eEF2K activity (). This activity was blocked when rottlerin was included in the reaction mixture, verifying the ability of this drug to inhibit this step. These data are consistent with previous reports where rottlerin failed to block the stimulatory effect of alcohol on eEF2 phosphorylation, even though it did inhibit the increased activity of eEF2K in response to AICAR, FBS or growth factors [Hong-Brown et al., 2007
; Parmer et al., 1997
; Parmer et al., 1999
]. Thus, these results indicate that there is an alternative mechanism that can control eEF2 activity, without the involvement of eEF2K. This conclusion is in agreement with published studies whereby exercise or treatment with farnesyltransferase induced inactivation of eEF2 in association with inhibition of protein synthesis [Ren et al., 2005
; Rose et al., 2005
]. These effects were also independent of the activity of eEF2K.
Previously, AMPK was reported to directly stimulate eEF2 phosphorylation following alcohol treatment [Hong-Brown et al., 2007
] and this response did not require the action of eEF2K. In the present study, AMPK was observed to activate eEF2K under in vitro conditions, and this activity increased in the presence of lopinavir. In addition, the lopinavir-induced increases in both eEF2K and eEF2 phosphorylation were blocked by the AMPK inhibitor compound C, suggesting that AMPK activates eEF2 via its effects on eEF2K. However, as stated above, data from our rottlerin experiments indicate that eEF2K is not required for this process. Moreover, AMPK was also shown to directly regulate eEF2 following lopinavir treatment (). Collectively, these data are consistent with the hypothesis that AMPK directly acts on eEF2, even when eEF2K and other upstream kinases pathways such as mTOR/S6K1 and ERK1/2 are inhibited (data not shown). Hence, in response to lopinavir, AMPK can regulate eEF2 in a manner that is independent of mTOR/eEF2K and ERK/eEF2K pathways.
Finally, we examined the role that phosphatases may play in regulating the effect of lopinavir on eEF2 phosphorylation. Previously, alcohol has been shown to decrease the PP2A activity against eEF2 [Hong-Brown et al., 2007
]. In contrast, lopinavir did not appear to inhibit PP2A activity. Although a role for other phosphatases can not be excluded, the observed changes in eEF2 phosphorylation were most likely due to changes in kinase activity.
We propose a model in which lopinavir increases the phosphorylation and activity of AMPK, thereby leading to an increased phosphorylation and inactivation of eEF2. Based on our in vivo and in vitro inhibitor studies, we suggest that AMPK can act on eEF2 either directly, or indirectly via the action of eEF2K. For example, treatment with the inhibitor compound C blocks the ability of AMPK to phosphorylate either eEF2K or eEF2, although it does not directly inhibit the activity of eEF2K towards eEF2. Likewise, rottlerin treatments block the activity of eEF2K towards eEF2, without affecting AMPK. In summary, these data are in agreement with the decrease in protein synthesis that occurs in myocytes exposed to lopinavir. As such, this should provide insight into the AMPK signaling mechanism regulating this process.