JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning.SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory.

A Protein Scaffold Coordinates SRC-Mediated JNK Activation in Response to Metabolic Stress.

Obesity is a major risk factor for the development of metabolic syndrome and type 2 diabetes. How obesity contributes to metabolic syndrome is unclear. Free fatty acid (FFA) activation of a non-receptor tyrosine kinase (SRC)-dependent cJun NH2-terminal kinase (JNK) signaling pathway is implicated in this process. However, the mechanism that mediates SRC-dependent JNK activation is unclear. Here, we identify a role for the scaffold protein JIP1 in SRC-dependent JNK activation. SRC phosphorylation of JIP1 creates phosphotyrosine interaction motifs that bind the SH2 domains of SRC and the guanine nucleotide exchange factor VAV. These interactions are required for SRC-induced activation of VAV and the subsequent engagement of a JIP1-tethered JNK signaling module. The JIP1 scaffold protein, therefore, plays a dual role in FFA signaling by coordinating upstream SRC functions together with downstream effector signaling by the JNK pathway.

JNK and PTEN cooperatively control the development of invasive adenocarcinoma of the prostate.

The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway is implicated in cancer, but the role of JNK in tumorigenesis is poorly understood. Here, we demonstrate that the JNK signaling pathway reduces the development of invasive adenocarcinoma in the phosphatase and tensin homolog (Pten) conditional deletion model of prostate cancer. Mice with JNK deficiency in the prostate epithelium (ΔJnk ΔPten mice) develop androgen-independent metastatic prostate cancer more rapidly than control (ΔPten) mice. Similarly, prevention of JNK activation in the prostate epithelium (ΔMkk4 ΔMkk7 ΔPten mice) causes rapid development of invasive adenocarcinoma. We found that JNK signaling defects cause an androgen-independent expansion of the immature progenitor cell population in the primary tumor. The JNK-deficient progenitor cells display increased proliferation and tumorigenic potential compared with progenitor cells from control prostate tumors. These data demonstrate that the JNK and PTEN signaling pathways can cooperate to regulate the progression of prostate neoplasia to invasive adenocarcinoma.

Requirement of JIP1-mediated c-Jun N-terminal kinase activation for obesity-induced insulin resistance.

The c-Jun NH(2)-terminal kinase (JNK) interacting protein 1 (JIP1) has been proposed to act as a scaffold protein that mediates JNK activation. However, recent studies have implicated JIP1 in multiple biochemical processes. Physiological roles of JIP1 that are related to the JNK scaffold function of JIP1 are therefore unclear. To test the role of JIP1 in JNK activation, we created mice with a germ line point mutation in the Jip1 gene (Thr(103) replaced with Ala) that selectively blocks JIP1-mediated JNK activation. These mutant mice exhibit a severe defect in JNK activation caused by feeding of a high-fat diet. The loss of JIP1-mediated JNK activation protected the mutant mice against obesity-induced insulin resistance. We conclude that JIP1-mediated JNK activation plays a critical role in metabolic stress regulation of the JNK signaling pathway.

Signal transduction cross talk mediated by Jun N-terminal kinase-interacting protein and insulin receptor substrate scaffold protein complexes.

Scaffold proteins have been established as important mediators of signal transduction specificity. The insulin receptor substrate (IRS) proteins represent a critical group of scaffold proteins that are required for signal transduction by the insulin receptor, including the activation of phosphatidylinositol 3 kinase. The c-Jun NH(2)-terminal kinase (JNK)-interacting proteins (JIPs) represent a different group of scaffold molecules that are implicated in the regulation of the JNK. These two signaling pathways are functionally linked because JNK can phosphorylate IRS1 on the negative regulatory site Ser-307. Here we demonstrate the physical association of these signaling pathways using a proteomic approach that identified insulin-regulated complexes of JIPs together with IRS scaffold proteins. Studies using mice with JIP scaffold protein defects confirm that the JIP1 and JIP2 proteins are required for normal glucose homeostasis. Together, these observations demonstrate that JIP proteins can influence insulin-stimulated signal transduction mediated by IRS proteins.

Role of the JIP4 scaffold protein in the regulation of mitogen-activated protein kinase signaling pathways.

The c-Jun NH2-terminal kinase (JNK)-interacting protein (JIP) group of scaffold proteins (JIP1, JIP2, and JIP3) can interact with components of the JNK signaling pathway and potently activate JNK. Here we describe the identification of a fourth member of the JIP family. The primary sequence of JIP4 is most closely related to that of JIP3. Like other members of the JIP family of scaffold proteins, JIP4 binds JNK and also the light chain of the microtubule motor protein kinesin-1. However, the function of JIP4 appears to be markedly different from other JIP proteins. Specifically, JIP4 does not activate JNK signaling. In contrast, JIP4 serves as an activator of the p38 mitogen-activated protein (MAP) kinase pathway by a mechanism that requires the MAP kinase kinases MKK3 and MKK6. The JIP4 scaffold protein therefore appears to be a new component of the p38 MAP kinase signaling pathway.

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice.

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.

The c-Abl tyrosine kinase phosphorylates the Fe65 adaptor protein to stimulate Fe65/amyloid precursor protein nuclear signaling.

The amyloid precursor protein (APP) is proteolytically processed to release a C-terminal domain that signals to the nucleus to regulate transcription of responsive genes. The APP C terminus binds to a number of phosphotyrosine binding (PTB) domain proteins and one of these, Fe65, stimulates APP nuclear signaling. Fe65 is an adaptor protein that contains a number of protein-protein interaction domains. These include two PTB domains, the second of which binds APP, and a WW domain that binds proline-rich ligands. One ligand for the Fe65WW domain is the tyrosine kinase c-Abl. Here, we show that active c-Abl stimulates APP/Fe65-mediated gene transcription and that this effect is mediated by phosphorylation of Fe65 on tyrosine 547 within its second PTB domain. The homologous tyrosine within the motif Tyr-(Leu/Met)-Gly is conserved in a variety of PTB domains, and this suggests that PTB tyrosine phosphorylation occurs in other proteins. As such, PTB domain phosphorylation may represent a novel mechanism for regulating the function of this class of protein.

The neuronal adaptor protein Fe65 is phosphorylated by mitogen-activated protein kinase (ERK1/2).

Fe65 is a neuronal adaptor protein that binds a number of ligands and which functions in both gene transcription/nuclear signalling and in the regulation of cell migration and motility. These different functions within the nucleus and at the cell surface are mediated via Fe65's different binding partners. An Fe65/APP/TIP60 complex is transcriptionally active within the nucleus and an Fe65/APP/Mena complex probably regulates actin dynamics in lamellipodia. The mechanisms that regulate these different Fe65 functions are unclear. Here, we demonstrate that Fe65 is a phosphoprotein and, using mass spectrometry sequencing, identify for the first time in vivo phosphorylation sites in Fe65. We also show that Fe65 is a substrate for phosphorylation by the mitogen-activated protein kinases ERK1/2. Our results provide a mechanism by which Fe65 function may be modulated to fulfil its various roles.

The neuronal adaptor protein X11alpha reduces Abeta levels in the brains of Alzheimer's APPswe Tg2576 transgenic mice.

Increased production and deposition of the 40-42-amino acid beta-amyloid peptide (Abeta) is believed to be central to the pathogenesis of Alzheimer's disease. Abeta is derived from the amyloid precursor protein (APP), but the mechanisms that regulate APP processing to produce Abeta are not fully understood. X11alpha (also known as munc-18-interacting protein-1 (Mint1)) is a neuronal adaptor protein that binds APP and modulates APP processing in transfected non-neuronal cells. To investigate the in vivo effect of X11alpha on Abeta production in the brain, we created transgenic mice that overexpress X11alpha and crossed these with transgenics harboring a familial Alzheimer's disease mutant APP that produces increased levels of Abeta (APPswe Tg2576 mice). Analyses of Abeta levels in the offspring generated from two separate X11alpha founder mice revealed a significant, approximate 20% decrease in Abeta(1-40) in double transgenic mice expressing APPswe/X11alpha compared with APPswe mice. At a key time point in Abeta plaque deposition (8 months old), the number of Abeta plaques was also deceased in APPswe/X11alpha mice. Thus, we report here the first demonstration that X11alpha inhibits Abeta production and deposition in vivo in the brain.

Cyclin-dependent kinase-5/p35 phosphorylates Presenilin 1 to regulate carboxy-terminal fragment stability.

Mutations in the Presenilin 1 gene are the cause of the majority of autosomal dominant familial forms of Alzheimer's disease. Presenilin 1 (PS1) is produced as a holoprotein but is then rapidly processed to amino- (N-PS1) and carboxy-terminal (C-PS1) fragments that are incorporated into stable high molecular mass complexes. The mechanisms that control PS1 cleavage and stability are not properly understood but sequences within C-PS1 have been shown to regulate both of these properties. Here we demonstrate that cyclin dependent kinase-5/p35 (cdk5/p35) phosphorylates PS1 on threonine(354) within C-PS1 both in vitro and in vivo. Threonine(354) phosphorylation functions to selectively stabilize C-PS1. Our results demonstrate that cdk5/p35 is a regulator of PS1 metabolism.