The transmembrane protein amyloid-β precursor protein (APP) and the vesicle-associated protein c-Jun NH2-terminal kinase–interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr668-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP–JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.

The amyloid-␤ (A␤) precursor protein (APP), a transmembrane protein that undergoes proteolytic cleavage into defined fragments, has been implicated in axonal transport. The proposed role of APP as a vesicle receptor for the microtubule motor kinesin-1 has relevance for the pathogenesis of Alzheimer's disease. Nevertheless, this function, which relies on the transport to the cell periphery of full-length APP rather than its cleavage fragments, remains controversial. Other proposed functions of APP, such as regulating transcription, neurogenesis, cell movement, or neurite growth also rely on APP's presence as a full-length protein at the cell surface, implying that APP cleavage occurs after its transport to the cell periphery. To test this hypothesis, we mapped the localization of various APP epitopes in neurons in culture and in the mouse brain. Surprisingly, epitopes from the N-terminal, C-terminal, and central (A␤) domains of APP each showed a distinct distribution throughout the cell and rarely colocalized. Within neurites, these epitopes were localized to distinct transport vesicles that associated with different sets of microtubules and, occasionally, actin filaments. C-terminal APP fragments were preferentially transported into neurites as phosphorylated forms, entered the lamellipodium and filopodia of growth cones, and concentrated in regions of growth cone turning and advancement (unlike the N-terminal and A␤ fragments). We conclude that, under normal conditions, the proteolytic cleavage of APP primarily occurs before its sorting into axonal transport vesicles and the cleaved fragments segregate into separate vesicle populations that reach different destinations, and thus have different functions.

Abnormal phosphorylation of amyloid-␤ precursor protein (APP) is a pathologic feature of Alzheimer's disease. To begin to understand the mechanism of APP phosphorylation, we studied this process in differentiating neurons under normal physiological conditions. We found that c-Jun NH 2 -terminal kinase (JNK), not cyclin-dependent kinase 5, is required for APP phosphorylation, leading to localized accumulation of phosphorylated APP (pAPP) in neurites. We show that JNK-interacting protein-3 (JIP-3), a JNK scaffolding protein that does not bind APP, selectively increases APP phosphorylation, accumulation of pAPP into processes, and stimulates process extension in both neurons and COS-1 cells. Downregulation of JIP-3 by small interfering RNA impairs neurite extension and reduces the amount of localized pAPP. Finally, whereas stress-activated JNK generates pAPP only in the cell body, concomitant expression of JIP-3 restores pAPP accumulation into neurites. Thus, APP phosphorylation, transport of the generated pAPP into neurites, and neurite extension are interdependent processes regulated by JIP-3/JNK, in a pathway distinct from stress-activated JNK signaling.

Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription-PCR for occludin-related sequences in Madin-Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction.

Beta-amyloid precursor protein (APP) is implicated in the pathobiology of Alzheimer's disease (AD). To gain insight into its function, we have investigated the proteolytic processing and post-translational modification of APP in relation to its intracellular traffic and localization. The proteolytic processing that generates the amyloid beta-peptide (Abeta) also releases into the cytoplasm the carboxy-terminal fragment of APP, Cgamma. Using the catecholaminergic cell line, CAD, and an antibody to a form of APP that is phosphorylated at Thr668 (pAPP; numbering for APP695), we show that a phosphorylated, carboxy-terminal fragment of APP, probably Cgamma, is present in the nucleus, where it localizes to subnuclear particles. The labeling with anti-pAPP antibody co-localizes with proteins that define the splicing factor compartment (SFC) [e.g. the small nuclear ribonucleoprotein (snRNP), U2B, and serine/arginine-rich (SR) proteins], but is excluded from the coiled bodies and the gems. This distribution of pAPP epitopes was found in CAD cells independent of their state of differentiation, as well as in primary cortical neurons, epithelial cells and fibroblasts. We further show that exogenously expressed Cgamma becomes phosphorylated, and distributes throughout the cell. A fraction of this Cgamma is translocated into the nucleus, where it co-localizes with endogenous pAPP epitopes. Finally, we show that the APP binding, scaffolding protein, Fe65 co-localizes with pAPP epitopes and with expressed Cgamma at intranuclear speckles. These results suggest that phosphorylated Cgamma accumulates at the SFC. Thus, APP may play a role in pre-mRNA splicing, and Fe65 and APP phosphorylation may regulate this function.