The potential role of glycogen synthase kinase-3␤ in modulating apoptosis was examined in human SH-SY5Y neuroblastoma cells. Staurosporine treatment caused time-and concentration-dependent increases in the activities of caspase-3 and caspase-9 but not caspase-1, increased proteolysis of poly(ADP-ribose) polymerase, and induced morphological changes consistent with apoptosis. Overexpression of glycogen synthase kinase-3␤ to levels 3.5 times that in control cells did not alter basal indices of apoptosis but potentiated staurosporine-induced activation of caspase-3, caspase-9, proteolysis of poly(ADP-ribose) polymerase, and morphological changes indicative of apoptosis. Inhibition of glycogen synthase kinase-3␤ by lithium attenuated the enhanced staurosporine-induced activation of caspase-3 in cells overexpressing glycogen synthase kinase-3␤. In cells subjected to heat shock, caspase-3 activity was more than three times greater in glycogen synthase kinase-3␤-transfected than control cells, and this potentiated response was inhibited by lithium treatment. Thus, glycogen synthase kinase-3␤ facilitated apoptosis induced by two experimental paradigms. These findings indicate that glycogen synthase kinase-3␤ may contribute to proapoptotic-signaling activity, that inhibition of glycogen synthase kinase-3␤ can contribute to anti-apoptotic-signaling mechanisms, and that the neuroprotective actions of lithium may be due in part to its inhibitory modulation of glycogen synthase kinase-3␤.Glycogen synthase kinase-3 (GSK-3) 1 was initially identified as a kinase that phosphorylates glycogen synthase (1). Subsequent studies have demonstrated that GSK-3 surpasses this function and plays a broad role in cellular metabolism, including contributions to signaling activities, growth, and differentiation (2). GSK-3␤ has been shown to phosphorylate numerous substrates, including several transcription factors such as c-jun, c-myc (3-5), and heat shock factor-1 (6), cytoskeletal proteins such as the microtubule-associated protein tau (7,8), and the multifunctional protein ␤-catenin (9). Thus it is now evident that the activity of GSK-3␤ influences a wide variety of cellular functions, including multiple signaling systems.Much still remains to be learned about the regulation of GSK-3␤ activity and its role as a modulator of signaling cascades that determine cell fate. Although often considered to be a constitutively active enzyme, GSK-3␤ can be both activated and inhibited. Activation has been shown to occur subsequent to phosphorylation of Tyr 216 (10) and recently by transient increases in intracellular calcium (11). Inhibition of GSK-3␤ can be induced by activation of the Wnt pathway (12) or by agents that activate a signaling cascade that commences when growth factors or insulin bind to their respective receptors (see Ref. 13 for review), resulting in the recruitment and activation of phosphatidylinositol 3-kinase. Activated phosphatidylinositol 3-kinase catalyzes the production of phosphatidylinositol 3,4,5-trisphosphate, which binds...

We describe here a new component of the phosphatidylinositol 3-kinase/Akt signaling pathway that directly impacts mitochondria. Akt (protein kinase B) was shown for the first time to be localized in mitochondria, where it was found to reside in the matrix and the inner and outer membranes, and the level of mitochondrial Akt was very dynamically regulated. Stimulation of a variety of cell types with insulin-like growth factor-1, insulin, or stress (induced by heat shock), induced translocation of Akt to the mitochondria within only several minutes of stimulation, causing increases of nearly eight-to 12-fold, and the mitochondrial Akt was in its phosphorylated, active state. Two mitochondrial proteins were identified to be phosphorylated following stimulation of mitochondrial Akt, the b-subunit of ATP synthase and glycogen synthase kinase-3b. The finding that mitochondrial glycogen synthase kinase-3b was rapidly and substantially modified by Ser9 phosphorylation, which inhibits its activity, following translocation of Akt to the mitochondria is the first evidence for a regulatory mechanism affecting mitochondrial glycogen synthase kinase-3b. These results demonstrate that signals emanating from plasma membrane receptors or generated by stress rapidly modulate Akt and glycogen synthase kinase-3b in mitochondria. Keywords: Akt, ATP synthase, glycogen synthase kinase3b, insulin-like growth factor-1, mitochondria, phosphatidylinositol 3-kinase. The serine/threonine kinase Akt (protein kinase B) plays a vital role in many cellular processes such as proliferation and survival (Lawlor and Alessi 2001). Akt can be activated by many types of stimuli (Datta et al. 1999), including growth factors, such as insulin-like growth factor-1 (IGF-1), hormones, such as insulin, and stressors, such as heat shock. Akt is most widely associated with the phosphatidylinositol 3-kinase (PI3K) signaling pathway where activation of Akt commences after PI3K catalyzes the production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate (Vanhaesebroeck and Alessi 2000). These lipids recruit Akt from the cytosol to the plasma membrane to facilitate the phosphorylation of Akt on Thr308 and Ser473 by phosphoinositide-dependent kinases (Datta et al. 1999;Vanhaesebroeck and Alessi 2000;Lawlor and Alessi 2001). The subsequent release of activated Akt from the membrane allows it to phosphorylate numerous substrates in the cytosol, and activated Akt also translocates into the nucleus Borgatti et al. 2000; BramiCherrier et al. 2002).One of the first identified substrates of Akt was glycogen synthase kinase-3b (GSK3b) (Cross et al. 1995). GSK3b, like Akt, affects many fundamental cellular functions, such as metabolism, survival, gene expression, and cytoskeletal dynamics, because of its ability to phosphorylate key proteins governing these processes (Grimes and Jope 2001). GSK3b is generally considered to be a constitutively active enzyme that is predominantly maintained in the cytosol, but both its activity and it...

Glycogen synthase kinase-3␤ (GSK3␤) is a central figure in Wnt signaling, in which its activity is controlled by regulatory binding proteins. Here we show that binding proteins outside the Wnt pathway also control the activity of GSK3␤. DNA damage induced by camptothecin, which activates the tumor suppressor p53, was found to activate GSK3␤. This activation occurred by a phosphorylation-independent mechanism involving direct binding of GSK3␤ to p53, which was confined to the nucleus where p53 is localized, and mutated p53 (R175H) bound but did not activate GSK3␤. Activation of GSK3 promoted responses to p53 including increases in p21 levels and caspase-3 activity. Thus, after DNA damage there is a direct interaction between p53 and GSK3␤, and these proteins act in concert to regulate cellular responses to DNA damage. C ells respond to DNA damage by activating signaling cascades that cause cell-cycle arrest to allow repair or cause apoptosis to eliminate irreparably damaged cells (1-3). After DNA damage, the tumor-suppressor protein p53 is a key intermediate in both cell-cycle arrest and apoptosis, and dysfunctional p53 is one of the most prevalent causes of tumor formation in humans (4-6). Apoptosis induced by p53 after DNA damage is executed by cysteine͞aspartate proteases such as the effector caspase-3, but regulatory mechanisms linking p53 to caspase activation remain unclear. Glycogen synthase kinase-3␤ (GSK3␤) is a key enzyme in several signaling pathways (7,8) including the Wnt pathway, through which its activity is controlled by regulatory binding proteins, and is an important proapoptotic signaling enzyme (9-12). Furthermore, apoptotic stimuli induce nuclear accumulation of GSK3␤ (13), colocalizing it with p53. Therefore, we examined whether there is a functional interaction between p53 and GSK3␤ and whether p53-mediated caspase activation caused by DNA damage involves GSK3␤. MethodsCell Culture and Treatments. Human neuroblastoma SH-SY5Y and H1299 cells were grown as described (11,14). Cells were washed and incubated in serum-free medium for 2 h before experimental treatments, and previously described procedures were used for preparation of cell lysates (13) and for measurements of caspase-3 activity (11). Full-length GSK3␤-binding protein (GBP) cDNA (provided by D. Kimelman) was amplified by PCR (primers 5Ј-AGT TAGTCGACGCCATGCCGTGTCG-CAAGGAG-3Ј and 5Ј-ACTATGCGGCCGCTTGCACGGTT-GTTCCAGT GCA-3Ј), digested with SalI and NotI, and inserted into the SalI͞NotI sites of the pCMV͞Myc͞nuc vector (Invitrogen) to make GBP with a nuclear localization signal and a Myc epitope. For construction of pcDNA3.1͞XG114, fulllength dominant negative GSK3␤ (provided by D. Kimelman) was excised from XG114 vector using BamHI and subcloned into the BamHI restriction site of pcDNA3.1(Ϫ) (Invitrogen). All constructs were verified by DNA sequencing. SH-SY5Y cell lines stably expressing nuclear localization signal-GBP or dominant negative GSK3␤ were generated as described (11).Immunoblot Analysis. Samples were mixed with Laemmli samp...

The recent discovery of direct interactions between two important regulators of cell fate, the tumor suppressor p53 and glycogen synthase kinase-3␤ (GSK3␤), led us to examine the mechanism and outcomes of this interaction. Two regions of p53 were identified that regulate its binding to GSK3␤. Deletion of the p53 activation domain-1 (AD1), but not mutations that prevent MDM2 binding through the AD1 domain, enhanced GSK3␤ binding to p53, indicating that the AD1 domain interferes with p53 binding to GSK3␤. Deletion of the p53 basic domain (BD) abrogated GSK3␤ binding, and a ten amino acid region within the C-terminal BD domain was identified as necessary for binding to GSK3␤. GSK3␤ activity was not required for p53 binding, but inhibition of GSK3␤ stabilized the association, suggesting a transient interaction during which active GSK3␤ promotes actions of p53. This regulatory role of GSK3␤ was demonstrated by large reductions of p53-induced increases in the levels of MDM2, p21, and Bax when GSK3␤ was inhibited. Besides promoting p53-mediated transcription, GSK3␤ also contributed to mitochondrial p53 apoptotic signaling. After DNA damage, mitochondrial GSK3␤ co-immunoprecipitated with p53 and was activated, and inhibition of GSK3␤ blocked cytochrome c release and caspase-3 activation. Thus, GSK3␤ interacts with p53 in both the nucleus and mitochondria and promotes its actions at both sites.

The goal of this study was to determine whether the intracellular distribution of the proapoptotic enzyme glycogen synthase kinase-3␤ (GSK-3␤) is dynamically regulated by conditions that activate apoptotic signaling cascades. In untreated human neuroblastoma SH-SY5Y cells, GSK-3␤ was predominantly cytosolic, although a low level was also detected in the nucleus. The nuclear level of GSK-3␤ was rapidly increased after exposure of cells to serum-free media, heat shock, or staurosporine. Although each of these conditions caused changes in the serine 9 and/or tyrosine phosphorylation of GSK-3␤, neither of these modifications was correlated with nuclear accumulation of GSK-3␤. Heat shock and staurosporine treatments increased nuclear GSK-3␤ prior to activation of caspase-9 and caspase-3, and this nuclear accumulation of GSK-3␤ was unaltered by pretreatment with a general caspase inhibitor. The GSK-3␤ inhibitor lithium did not alter heat shock-induced nuclear accumulation of GSK-3␤ but increased the nuclear level of cyclin D1, indicating that cyclin D1 is a substrate of nuclear GSK-3␤. Thus, the intracellular distribution of GSK-3␤ is dynamically regulated by signaling cascades, and apoptotic stimuli cause increased nuclear levels of GSK-3␤, which facilitates interactions with nuclear substrates.