Claire L. Standen

Phosphorylation of thr668 in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3)

Threonine668 (thr668) within the carboxy‐terminus of the Alzheimers disease amyloid precursor protein (APP) is a known in vivo phosphorylation site. Phosphorylation of APPthr668 is believed to regulate APP function and metabolism. Thr668 precedes a proline, which suggests that it is targeted for phosphorylation by proline‐directed kinase(s). We have investigated the ability of four major neuronally active proline‐directed kinases, cyclin dependent protein kinase‐5, glycogen synthase kinase‐3β, p42 mitogen‐activated protein kinase and stress‐activated protein kinase‐1b, to phosphorylate APPthr668 and report here that SAPK1b induces robust phosphorylation of this site both in vitro and in vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.

X11 alpha and x11 beta interact with presenilin-1 via their PDZ domains.

Abstract X11α and X11β are two neuronal adaptor proteins that interact with the Alzheimers disease amyloid precursor protein (APP). X11α and X11β stabilise APP and inhibit production of proteolytic APP fragments including the Aβ peptide that is deposited in the brains of Alzheimers disease patients. The mechanisms by which X11α and X11β modulate APP processing are not clear but one possibility is that they influence the activity of the secretases that cleave APP to give rise to Aβ. Presenilin-1 is required for γ-secretase activity and here we demonstrate that both X11α and X11β interact with presenilin-1. X11/presenilin-1 binding is via two X11 PDZ domains and sequences within the carboxy-terminus of presenilin-1. We also demonstrate that both X11α and X11β mediate the formation of complexes between APP and presenilin-1. These results suggest that the X11 regulation of APP processing is controlled, at least in part, via their interactions with APP and presenilin-1.

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.

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 Alzheimers 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.

The neuronal adaptor protein X11alpha interacts with the copper chaperone for SOD1 and regulates SOD1 activity.

The neuronal adaptor protein X11α participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human X11α, and we isolated a clone encoding domains II and III of the copper chaperone for Cu,Zn-superoxide dismutase-1 (CCS). The X11α/CCS interaction was confirmed in coimmunoprecipitation studies plus glutathioneS-transferase fusion protein pull-down assays and was shown to be mediated via PDZ2 of X11α and a sequence within the carboxyl terminus of domain III of CCS. CCS delivers the copper cofactor to the antioxidant superoxide dismutase-1 (SOD1) enzyme and is required for its activity. Overexpression of X11α inhibited SOD1 activity in transfected Chinese hamster ovary cells which suggests that X11α binding to CCS is inhibitory to SOD1 activation. X11α also interacts with another copper-binding protein found in neurons, the Alzheimers disease amyloid precursor protein. Thus, X11α may participate in copper homeostasis within neurons.

Fe65 and X11beta co-localize with and compete for binding to the amyloid precursor protein.

The Fe65s and X11s are two families of adaptor proteins that bind to the Alzheimers disease amyloid precursor protein (APP). Although both the X11s and Fe65s bind to similar regions of APP, they have opposing effects on Abeta production and hence may represent novel therapeutic targets. However, there is no evidence that the Fe65s and X11s are present within the same cell type or cell compartment and are thus capable of competing for binding to APP. Here we show that in neurones and transfected cells, APP, Fe65 and X11beta show overlapping subcellular distributions. Furthermore, we demonstrate that Fe65 and X11beta compete for binding to APP.

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 Fe65s 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.