Glycogen synthase kinase-3 (GSK3) is implicated in the regulation of several physiological processes, including the control of glycogen and protein synthesis by insulin, modulation of the transcription factors AP-1 and CREB, the specification of cell fate in Drosophila and dorsoventral patterning in Xenopus embryos. GSK3 is inhibited by serine phosphorylation in response to insulin or growth factors and in vitro by either MAP kinase-activated protein (MAPKAP) kinase-1 (also known as p90rsk) or p70 ribosomal S6 kinase (p70S6k). Here we show, however, that agents which prevent the activation of both MAPKAP kinase-1 and p70S6k by insulin in vivo do not block the phosphorylation and inhibition of GSK3. Another insulin-stimulated protein kinase inactivates GSK3 under these conditions, and we demonstrate that it is the product of the proto-oncogene protein kinase B (PKB, also known as Akt/RAC). Like the inhibition of GSK3 (refs 10, 14), the activation of PKB is prevented by inhibitors of phosphatidylinositol (PI) 3-kinase.

In the three decades since pluripotent mouse embryonic stem (ES) cells were first described 1,2 they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts [1][2][3][4][5][6][7] . Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.Reprints and permissions information is available at www.nature.com/reprints.Correspondence and requests for materials should be addressed to Q.L.Y. (qying@keck.usc.edu) or A.S. (ags39@cscr.cam.ac.uk). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. We found that, in combination with LIF, either inhibitor replaces the requirement for serum/BMP and supports robust long-term ES-cell propagation (Supplementary Information). Lineage commitment does not occur despite a reduced expression of inhibitorof-differentiation proteins. In contrast, ES cells plated without LIF in either PD184352 or SU5402 progressively degenerate and cannot be maintained even though differentiation is suppressed. To reduce off-target side effects we tried low doses of PD184352 and SU5402 together (PS). In PS we find that undifferentiated ES cells expand through multiple passages (Fig. 1a, b). Differentiation is constrained, although occasional neural rosettes emerge. This result, observed with several independent ES cell lines, suggests that the minimal requirements for ES-cell self-renewal may be to deflect commitment signals emanating from FGF receptor and ERK signalling. However, apoptosis is relatively high in PS, especially immediately after passage, and cells survive poorly at clonal density, which is indicative of collateral compromise to cell growth and viability. Author ContributionsES-cell propagation has been reported to be enhanced by an indirubin entity, 6-bromoindirubin-3′-oxime (BIO), that inhibits glycogen synthase kinase-3 (GSK3) 4 . ...

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.