Mcl-1 is normally subject to rapid up- and downregulation, to modulate cell viability in response to environmental signals and maintain tissue homeostasis 
. Mcl-1 can be targeted for degradation by several pathways, some of which are regulated post-translationally. Phosphorylation at Thr 163 was initially observed in TPA-treated BL41-3 cells, where it is associated with Mcl-1 stabilization 
. Thr 163 phosphorylation also occurs in UV-irradiated mouse fibroblasts where it primes for GSK3-induced Ser 159 phosphorylation and Mcl-1 degradation 
. In, the present work, this pathway was not found to play a major role in Mcl-1 degradation in BL41-3 cells. These cells may thus provide a model for the many types of cancer cells that exhibit impaired Mcl-1 degradation through the GSK3-targeted pathway 
. The finding that Thr 163 phosphorylation can promote Mcl-1 stabilization in this situation in BL41-3 cells was mimicked in CHO cells transfected with phosphorylation site mutants. Overall, Thr 163 phosphorylation can prime GSK3-targeted Mcl-1 degradation to promote death in normal cells; however, in cancer cells in which degradation is not dependent on this pathway, ERK activation and the induction of Thr 163 phosphorylation are associated with marked Mcl-1 stabilization and chemotherapeutic drug resistance.
Whereas Thr 163 phosphorylation in BL41-3 cells is induced by ERK, the priming phosphorylation in mouse fibroblasts is carried out by JNK, activated upon UV irradiation. Therefore, additional events induced by JNK may influence GSK3/phosphoSer 159 mediated Mcl-1 degradation in this system. Further studies of non-transformed hematopoietic cell lines may be informative in this regard. In the latter, Thr 163 phosphorylation is thought to prime the GSK3-induced Ser 159 phosphorylation/Mcl-1 degradation that occurs following growth factor deprivation 
. However, whether ERK or JNK is involved has not yet been investigated. Taken together, findings from various systems suggest that MAP kinase (ERK or JNK)-induced phosphorylation at Thr 163 can either prime for Mcl-1 degradation – in cells in which this is targeted by GSK3-induced Ser 159 phosphorylation – or have the opposite effect to stabilize Mcl-1 in cells in which degradation is carried out by other, GSK3-in
Abundant, dysregulated Mcl-1 expression is an important determinant of drug resistance in cancer 
. This often involves alterations affecting GSK3-targeted Mcl-1 degradation, such as GSK3 inactivation or changes in downstream components in the pathway 
. However, it is not clear why reduced Mcl-1 degradation through one pathway provides a substantial advantage to tumor cells, since multiple alternative pathways can target Mcl-1 degradation and might be expected to provide at least partial compensation. In the case of inactivation of the F-box protein FBW7, for example, Mcl-1 degradation is slowed but still occurs relatively rapidly 
. The present findings provide food for thought in this regard as they show that – in cells in which Mcl-1 degradation does not depend on the GSK3/phosphodegron – another common event in cancer, ERK activation, is associated with Thr 163 phosphorylation, Mcl-1 stabilization, and a dramatic increase in drug resistance. These findings also point to the importance of future studies aimed at assessing whether Mcl-1 Thr 163 phosphorylation could serve as a resistance marker in cancer patients.
The various events induced by TPA in BL41-3 cells appear to be closely linked. TPA-induced ERK activation, Thr 163 phosphorylation, and Mcl-1 stabilization occur as early, U126-inhibitable events and are subsequently downregulated. Similarly, upon exposure to chemotherapeutic drugs, TPA-induced inhibition of Mcl-1 degradation and apoptosis are seen at early times and exhibit a close correspondence. Because of this, a cause/effect relationship between these events could not be distinguished. Likewise, causality could not be meaningfully addressed via transfection with Mcl-1-T163E, because the extensive stability of the mutant protein resulted in its build-up to levels that far exceeded those seen physiologically. We also note that TPA has a multitude of effects, and other effects besides ERK activation, Thr 163 phosphorylation, and Mcl-1 stabilization may contribute to the drug resistance observed in BL41-3 cells. Whatever the case, a variety of chemotherapeutic drugs, applied at a range of doses, uniformly caused a decrease in Mcl-1 expression that correlated with the increase in PARP cleavage (approximately a 1
1 correlation), both events being inhibited upon TPA-induced ERK activation and Thr 163 phosphorylation. Thus, effects on Mcl-1 expression may contribute to, and serve as a surrogate marker for, effects on drug sensitivity/resistance.
A further observation related to those above came out of studies of TPA-induced viability-protection in cells treated with chemotherapeutic agents for 24 hours. While the direct effects of TPA on ERK activation, Thr 163 phosphorylation, and Mcl-1 stabilization – in the absence of chemotherapeutic drugs - were downregulated at this time, cell exposed to chemotherapeutic agents for 24 hours exhibited less apoptosis in the presence (versus the absence) of TPA. The maintenance of Mcl-1 expression and increased survival at early times may allow more cells to remain viable upon prolonged exposure. In terms of patient cancers in which GSK3-mediated Mcl-1 degradation is inactive, it remains to be determined whether the presence of ERK activation/Thr 163 phosphorylation contributes to resistance upon prolonged drug exposure.
It was also interesting that U0126 partially reversed the protective effects of TPA. Why reversal was only partial is not clear, since the inhibitor has been found to be highly effective in terms of minimizing TPA-induced ERK activation. One possibility is that other ERK-independent effects of TPA play a role, and another possibility is that some ERK activation occurs in the presence of U0126. Examination of pERK expression did not show detectable ERK activation in the presence of U1026 plus TPA, as in previous studies 
. However, trace pERK appeared to be visible in the presence of these two agents plus etoposide (not shown). In previous studies, we have observed that a modicum of ERK activation, even if it is transient, can have a noticeable effect 
. TPA is a powerful ERK inducer and it remains to be determined whether some activation, possibly transient, occurs in the present system. U0126 did not substantially counteract TPA-induced resistance at a low concentration of etoposide (3.3 micromolar), but promoted apoptosis in the presence of higher etoposide concentrations. In other words, in cancer cells exhibiting drug resistance in the presence of ERK activation and GSK3 inactivation, an effective approach may involve inhibition of the effects of ERK along with the application of full doses of chemotherapeutic agents.
Mcl-1 is highly regulated at multiple levels, and ERK-induced transcriptional and post-translational mechanisms have been suggested to act coordinately to rapidly increase or decrease expression 
. The present findings lead us to speculate that coordination may also exist for pathways that degrade the protein. For example, when Mcl-1 degradation via the GSK3-targeted phosphodegron is downregulated, such as upon growth factor-stimulation 
, ERK-induced Thr 163 phosphorylation may provide a means for preventing degradation via alternative routes. This could avert a situation in which the GSK3-targeted pathway is downregulated, but this does not have a strong impact because other degradation pathways remain fully operational. In addition, as the growth stimulus wanes and GSK3 activation increases, the presence of Thr 163 phosphorylation may prime for ensuing Mcl-1 degradation. In other words, this could also provide a mechanism for preventing overly sustained Mcl-1 expression by coupling growth factor-induced Mcl-1 stabilization to priming for subsequent turnover. The interplay between these factors may then be exploited by cancer cells, where activation of ERK and inactivation of GSK3/phosphodegron-mediated degradation would promote extended Mcl-1 stabilization and drug resistance.