In this report, we provide evidence that S6K2 plays a role in IL-3-mediated cell signaling. S6K2 is activated when Ba/F3 cells or primary mouse bone marrow-derived mast cells are stimulated with IL-3, and this signaling is mediated via the PI-3K and mTOR pathway but not by the MEK pathway in Ba/F3 cells. In fibroblasts, it has been shown that the MEK pathway plays a role in activating S6K2; S6K2 activity, which is induced by serum, EGF, or FGF-2, is sensitive to treatment with U0126 [16
]. It is thought that serines 410, 417, and 423 in the C terminus of S6K2 play a role in autoinhibition by acting as a pseudosubstrate in quiescence, and phosphorylation of these sites upon mitogenic stimulation relieves autoinhibitory effects and initiates activation of S6K2 [16
]. When these serines are mutated to aspartic acid, S6K2 becomes insensitive to the inhibitory effects of U0126 when stimulated with EGF or serum [16
]. We observe in Ba/F3 cells that U0126 does not have inhibitory effects on S6K2 activity, which suggests that a mitogenic signal different from the MEK pathway contributes to phosphorylation and lift in autoinhibition of S6K2. Indeed, Martin et al. [16
] have shown that U0126 inhibits activation of S6K2 by EGF but not insulin in the same cell line, HEK293, indicating that different growth factor receptors use different mechanisms even within the same cell.
It is interesting to note that T388E S6K2 has been shown to be constitutively active in HEK293 cells in the absence of serum [14
]. However, we did not observe constitutive catalytic activity of T388E when it was expressed in Ba/F3 cells in the absence of IL-3, although the IL-3-stimulated catalytic activity was still rapamycin-resistant. This suggests that different regulation of S6K2 catalytic activity occurs in the two cell types. One possible mechanism is that there exists in HEK293 cells a constitutively active signal to S6K2 that renders it partly ready to be activated, and with only addition of the T388E mutation, S6K2 can become fully active in the absence of serum. In Ba/F3 cells, this putative signal may not be constitutive but instead active only in the presence of IL-3.
Ba/F3 cells expressing T388E proliferate better at all doses of IL-3; yet, in the absence of IL-3, the cells fail to proliferate. This suggests that expressing T388E in itself is not sufficient to drive proliferation and/or survival of Ba/F3. This is consistent with the fact that IL-3 activates other signaling pathways known to contribute to proliferation and survival, including the JAK/STAT pathway, which are divergent from the mTOR pathway and need to be activated along with mTOR. Proliferation of T388E-expressing Ba/F3 cells was inhibited by rapamycin in a dose-dependent manner, although these cells still proliferate better than the control cells at all doses of rapamycin. The observation that expressing T388E in Ba/F3 cells is not sufficient to overcome inhibition of proliferation by rapamycin indicates that other molecule(s), which are downstream of mTOR, play an important role in IL-3-mediated cell proliferation and that T388E cannot substitute for their activity. We envision these molecule(s) to be parallel to S6K2 in the mTOR pathway. When rapamycin is added to cells, although S6K2 activity is overexpressed and may give some proliferative advantage, these other molecule(s) would still be inhibited by rapamycin, resulting in inhibition of proliferation.
How does T388E mediate a proliferative advantage in the presence of IL-3? There are two possibilities. T388E may influence IL-3-mediated signaling by partly activating one of the signaling pathways necessary for cell-cycle progression, thereby allowing easier activation and proliferation when IL-3 is added. In other words, T388E may influence the IL-3 signaling pathway in a quantitative manner. Alternatively, T388E may affect the IL-3 signaling pathway in a qualitative manner, by changing the way cells behave in response to proliferation signals. If T388E affects the proliferative response by partly activating one of the signaling pathways (i.e., in a quantitative manner), it could be predicted that at a maximal dose of IL-3, higher cell numbers in T388E-expressing cells would not necessarily be observed, as all signaling pathways would be activated maximally at that dose. However, T388E-expressing cells proliferate better at all IL-3 doses, including at a maximal dose, which suggests that T388E changes qualitatively the way Ba/F3 cells proliferate. One possibility is that T388E-expressing cells go through the cell cycle at a faster rate, resulting in greater cell numbers. Indeed, when we assessed the cell-cycle profile of cells expressing T388E, we observed that these cells enter S phase earlier. These findings suggest that S6K2 may play a role in G1-phase length regulation and/or S-phase entry. Rapamycin blocks or delays cell-cycle progression from G1 to S phase, and it has recently been shown that U2OS cells expressing a rapamycin-resistant form of mTOR can bypass a rapamycin-induced S-phase entry block when stimulated with serum [25
]. This was partly mediated through S6K1 and 4E-BP1, as overexpressing eIF4E or a rapamycin-resistant form of S6K1 exhibited modest but significant acceleration in S-phase entry [25
]. The study did not address whether S6K2 also plays a role in mTOR-mediated S-phase entry. Here, we present evidence that S6K2 can also play a role in S-phase entry in Ba/F3 cells proliferating with IL-3 stimulation. We do not rule out the possibility that there may exist other mechanisms by which IL-3-mediated cell proliferation is aided by expression of T388E. IL-3 in hematopoietic cells promotes not only cell proliferation but also cell survival by regulating apoptotic-signaling pathways, most notably by up-regulating expression of Bcl-x and Bcl-2 and by inhibiting BAD through phosphorylation [1
]. It has also been reported that an S6K2-related kinase, S6K1, can phosphorylate BAD and may play a role in apoptosis [26
]. Substrates for S6K2, other than S6, have not been found to date. Further examination about whether S6K2 plays a role in apoptosis may yield better insights into the mechanism of S6K2-mediated proliferation regulation.
It is not clear whether the T388E-mediated proliferative advantage is the result of increased phosphorylation of S6 or other substrates. S6K2 was first identified via its homology to S6K1, an in vivo kinase for S6. A S6K1/S6K2 double-knockout mouse study shows that cells derived from the double null mice have virtually no S6 phosphorylation, whereas cells that lack one or the other kinase have only partially reduced S6 phosphorylation, indicating that S6 is likely to be an in vivo substrate of S6K2 [19
]. Our data show that S6 is phosphorylated in a rapamycin-resistant manner in cells expressing T388E, establishing that T388E behaves as an in vivo S6 kinase in this system as well. However, S6K2 might have other substrates relevant to cell-cycle progression. Indeed, recently an S6K1-specific substrate, SKAR, was identified, indicating that S6K1 and S6K2 may have nonoverlapping subsets of substrates as well as the shared substrate S6 [21
]. Studies are currently under way for identifying the protein(s) whose phosphorylation is affected when S6K2 is diminished in or near S phase, with the purpose of elucidating the mechanism of S-phase entry regulation by S6K2.
In summary, we have shown that IL-3-mediated cell proliferation is potentiated by S6K2 and that S6K2 may play a role in regulating G1-phase duration and/or S-phase entry. Finding a full spectrum of S6K2 substrates in G1 phase may yield better insights to the mechanism of S6K2-mediated G1/S-phase regulation.