Rapid and bi-directional regulation of AMPA receptor phosphorylation and trafficking by JNK

Jun N-terminal kinases (JNKs) are implicated in various neuropathological conditions. However, physiological roles for JNKs in neurons remain largely unknown, despite the high expression level of JNKs in brain. Here, using bioinformatic and biochemical approaches, we identify the AMPA receptor GluR2L and GluR4 subunits as novel physiological JNK substrates in vitro, in heterologous cells and in neurons. Consistent with this finding, GluR2L and GluR4 associate with specific JNK signaling components in the brain. Moreover, the modulation of the novel JNK sites in GluR2L and GluR4 is dynamic and bi-directional, such that phosphorylation and de-phosphorylation are triggered within minutes following decreases and increases in neuronal activity, respectively. Using live-imaging techniques to address the functional consequence of these activity-dependent changes we demonstrate that the novel JNK site in GluR2L controls reinsertion of internalized GluR2L back to the cell surface following NMDA treatment, without affecting basal GluR2L trafficking. Taken together, our results demonstrate that JNK directly regulates AMPA-R trafficking following changes in neuronal activity in a rapid and bi-directional manner.     

Positive and negative regulation of Raf kinase activity and function by phosphorylation.

Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function.     

Positive and negative regulation of insulin signaling through IRS-1 phosphorylation

This review will provide insight on the current understanding of the regulation of insulin signaling in both physiological and pathological conditions through modulations that occur with regards to the functions of the insulin receptor substrate 1 (IRS1). While the phosphorylation of IRS1 on tyrosine residue is required for insulin-stimulated responses, the phosphorylation of IRS1 on serine residues has a dual role, either to enhance or to terminate the insulin effects. The activation of PKB in response to insulin propagates insulin signaling and promotes the phosphorylation of IRS1 on serine residue in turn generating a positive-feedback loop for insulin action. Insulin also activates several kinases and these kinases act to induce the phosphorylation of IRS1 on specific sites and inhibit its functions. This is part of the negative-feedback control mechanism induced by insulin that leads to termination of its action. Agents such as free fatty acids, cytokines, angiotensin II, endothelin-1, amino acids, cellular stress and hyperinsulinemia, which induce insulin resistance, lead to both activation of several serine/threonine kinases and phosphorylation of IRS1. These agents negatively regulate the IRS1 functions by phosphorylation but also via others molecular mechanisms (SOCS expression, IRS degradation, O-linked glycosylation) as summarized in this review. Understanding how these agents inhibit IRS1 functions as well as identification of kinases involved in these inhibitory effects may provide novel targets for development of strategies to prevent insulin resistance.     

Angiotensin II stimulates rapid serine phosphorylation of transcription factor Stat3

Lipid peroxidation (LPO) in rat testis and heart microsomes was compared using the ADP/Fe2+ as initiator with and without ascorbate at different concentrations. The extent of LPO was estimated by the levels of TBARS and PUFA. Without ascorbate, LPO was higher in heart than in testis despite elevated levels of catalase in heart. With increased ascorbate concentrations, a biphasic effect of LPO was observed. For a concentration 0.2 mM, ascorbate acted as pro-oxidant and increased TBARS correlated with decreased PUFA were observed both in testis and heart. Above 0.2 mM, ascorbate acts as antioxidant but differences in the rate of LPO were observed. In heart decreased TBARS correlated with increased PUFA whereas in testis TBARS only decreased, PUFA were not significantly modified. These results suggest different mechanisms in LPO initiation in the two organs. Increasing concentrations of H2O2 produced directly elevated TBARS levels in testis while a lag phase was observed in heart before the increase, suggesting that H2O2 was the essential ROS produced by ascorbate-ADP/Fe2+. The effects of scavengers such as catalase and ethanol showed an inhibitory effect on TBARS production only in testis, suggesting the role of H2O2/OH as an initiator of LPO. In heart, catalase produced a slight increase in TBARS levels whereas no modification was observed with ethanol, suggesting a possible direct activation by ADP/Fe2+ through a metal-oxo intermediate.     

Dual role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation

Autophagy and apoptosis are fundamental cellular pathways that are both regulated by JNK-mediated Bcl-2 phosphorylation. Several years ago, JNK-mediated Bcl-2 phosphorylation was shown to interfere with its binding to proapoptotic BH3 domain-containing proteins such as Bax and recently, our laboratory demonstrated that JNK1-mediated Bcl-2 phosphorylation interferes with its binding to the proautophagy BH3 domain-containing protein Beclin 1. Here, we examined the kinetic relationship between Bcl-2 phosphorylation, Bcl-2-Beclin 1 interactions, Bcl-2-Bax interactions, and caspase 3 activation during nutrient starvation. We found that after a short period of nutrient deprivation (4 hours), a small amount of Bcl-2 phosphorylation dissociates Bcl-2 from the Bcl-2-Beclin 1 complex but not from the Bcl-2-Bax complex. After 16 hours of nutrient deprivation, Bcl-2 phosphorylation reaches maximal levels, the Bcl-2-Bax complex is disrupted, and active caspase 3 is detected, indicating the initiation of apoptosis. Based on this result, we propose a speculative model for understanding the interrelationship between autophagy and apoptosis regulated by JNK1-mediated Bcl-2 phosphorylation. According to this model, rapid Bcl-2 phosphorylation may occur initially to promote cell survival by disrupting the Bcl-2-Beclin 1 complex and activating autophagy. At a certain point when autophagy is no longer able to keep the cell alive, Bcl-2 phosphorylation might then serve to inactivate its antiapoptotic function.     

Dual Role of JNK1-mediated phosphorylation of Bcl-2 in autophagy and apoptosis regulation

Autophagy and apoptosis are fundamental cellular pathways that are both regulated by JNK-mediated Bcl-2 phosphorylation. Several years ago, JNK-mediated Bcl-2 phosphorylation was shown to interfere with its binding to pro-apoptotic BH3 domain-containing proteins such as Bax and recently, our laboratory demonstrated that JNK1-mediated Bcl-2 phosphorylation interferes with its binding to the pro-autophagy BH3 domain-containing protein Beclin 1. Here, we examined the kinetic relationship between Bcl-2 phosphorylation, Bcl-2-Beclin 1 interactions, Bcl-2-Bax interactions and caspase 3 activation during nutrient starvation. We found that after a short period of nutrient deprivation (4 hours), a small amount of Bcl-2 phosphorylation dissociates Bcl-2 from the Bcl-2-Beclin 1 complex but not from the Bcl-2-Bax complex. After 16 hours of nutrient deprivation, Bcl-2 phosphorylation reaches maximal levels, the Bcl-2-Bax complex is disrupted, and active caspase 3 is detected, indicating the initiation of apoptosis. Based on this result, we propose a speculative model for understanding the interrelationship between autophagy and apoptosis regulated by JNK1-mediated Bcl-2 phosphorylation. According to this model, rapid Bcl-2 phosphorylation may occur initially to promote cell survival by disrupting the Bcl-2-Beclin 1 complex and activating autophagy. At a certain point when autophagy is no longer able to keep the cell alive, Bcl-2 phosphorylation might then serve to inactivate its anti-apoptotic function.

Addendum to: Wei Y, Pattingre S, Sinha S, Bassik M, Levine B. JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Mol Cell 2008; 30:678-88.     

A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a +TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure.     

Serine 332 phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 attenuates insulin signaling

The ability of glycogen synthase kinase-3 (GSK-3) to phosphorylate insulin receptor substrate-1 (IRS-1) is a potential inhibitory mechanism for insulin resistance in type 2 diabetes. However, the serine site(s) phosphorylated by GSK-3 within IRS-1 had not been yet identified. Using an N-terminal deleted IRS-1 mutant and two IRS-1 fragments, PTB-1 1-320 and PTB-2 1-350, we localized GSK-3 phosphorylation site(s) within amino acid sequence 320-350. Mutations of serine 332 or 336, which lie in the GSK-3 consensus motif (SXXXS) within PTB-2 or IRS-1, to alanine abolished their phosphorylation by GSK-3. This suggested that Ser332 is a GSK-3 phosphorylation site and that Ser336 serves as the "priming" site typically required for GSK-3 action. Indeed, dephosphorylation of IRS-1 prevented GSK-3 phosphorylation. Furthermore, the phosphorylated peptide derived from the IRS-1 sequence was readily phosphorylated by GSK-3, in contrast to the nonphosphorylated peptide, which was not phosphorylated by the enzyme. When IRS-1 mutants S332A(IRS-1), S336A(IRS-1), or S332A/336A(IRS-1) were expressed in Chinese hamster ovary cells overexpressing insulin receptors, their insulin-induced tyrosine phosphorylation levels increased compared with that of wild-type (WT) IRS-1. This effect was stronger in the double mutant S332A/336A(IRS-1) and led to enhanced insulin-mediated activation of protein kinase B. Finally, immunoblot analysis with polyclonal antibody directed against IRS-1 phosphorylated at Ser332 confirmed IRS-1 phosphorylation in cultured cells. Moreover, treatment with the GSK-3 inhibitor lithium reduced Ser332 phosphorylation, whereas overexpression of GSK-3 enhanced this phosphorylation. In summary, our studies identify Ser332 as the GSK-3 phosphorylation target in IRS-1, indicating its physiological relevance and demonstrating its novel inhibitory role in insulin signaling.     

JNK regulation of oncogenesis

The literature provides strong precedent for both pro-tumorigenic and tumor suppressor roles for the c-Jun N-terminal kinases (JNKs) in the setting of oncogenesis. Clearly, JNKs are activated by numerous oncogenes and growth factors and the literature documents a role for these MAP kinases in cell proliferation and transformation. By contrast, JNKs mediate signals from diverse stimuli that result in cell death or differentiation and a role for JNKs as tumor suppressors has emerged. This enigmatic nature of the JNKs in the setting of oncogenesis is considered herein. Further illumination of the complex and context-dependent functions of the JNKs in cancer cells is of obvious importance for the rational use of small molecule JNK inhibitors as therapeutics.