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Human Molecular Genetics, 2004, Vol. 13, No. 5 475-488
DOI: 10.1093/hmg/ddh054

A phosphorylated, carboxy-terminal fragment of ß-amyloid precursor protein localizes to the splicing factor compartment

Zoia Muresan and Virgil Muresan*

Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4970, USA

Received September 5, 2003; Revised December 15, 2003; Accepted January 2, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-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 ß-peptide (Aß) also releases into the cytoplasm the carboxy-terminal fragment of APP, C{gamma}. 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 C{gamma}, 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 C{gamma} becomes phosphorylated, and distributes throughout the cell. A fraction of this C{gamma} 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 C{gamma} at intranuclear speckles. These results suggest that phosphorylated C{gamma} accumulates at the SFC. Thus, APP may play a role in pre-mRNA splicing, and Fe65 and APP phosphorylation may regulate this function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Alzheimer's disease (AD) is a neurodegenerative disorder of unknown etiology (1). A large body of evidence implicates ß-amyloid precursor protein (APP), a ubiquitously expressed, type I transmembrane protein, in the pathobiology of AD (2). Neuritic plaques, one of the neuropathological lesions observed in the brains of AD patients, contain extracellular deposits of amyloid ß-peptide (Aß) generated by proteolytic processing of APP (1). In spite of the strong correlation between AD and APP metabolism, the function of APP itself is still unclear. This study addresses the biological role of APP.

APP is synthesized in the endoplasmic reticulum, becomes processed by glycosylation and sulfation in the Golgi complex, and is exported to the plasma membrane in small transport vesicles (2,3). A fraction of the APP that reaches the plasma membrane re-enters the cell via endocytosis. Somewhere along this pathway, APP is processed, through the action of ß- and {gamma}-secretase, by proteolytic cleavage at two positions, one of which is an intra-membrane site. Aside from generating the topologically extracellular Aß and a large amino-terminal fragment, secretase cleavage of APP also releases into the cytoplasm a carboxy-terminal fragment, C{gamma} (reviewed in 2,4).

Recently, it has been suggested that APP signals to the nucleus in a manner analogous to other transmembrane signaling proteins that undergo {gamma}-secretase mediated, intramembrane proteolysis (reviewed in 5), such as the cell surface receptor Notch (6), the cell–cell adhesion molecule, E-cadherin (7), the receptor tyrosine kinase ErbB-4 (8), and the low density lipoprotein receptor-related protein (LRP) (9). In the case of Notch, this process is initiated by the interaction with its ligand, Delta. The released Notch intracellular domain interacts with the cellular factor CSL and translocates into the nucleus where it modifies gene transcription (reviewed in 5). By analogy, it was proposed that the released intracellular domain of APP, C{gamma}, is transported to the nucleus in a complex with the adaptor protein Fe65, where it may alter gene expression (10,11). The hypothesis that APP plays a role in regulating transcription is based on data showing that C{gamma} translocates into the nucleus when co-expressed with Fe65 (12,13), and on studies using heterologous signal transduction assays (11,14). While regulating transcription is one possible function of C{gamma}, its role in the nucleus is still unclear.

The mature, N- and O-glycosylated form of APP695 (i.e. the major neuronal isoform of APP) is phosphorylated at Thr668 (15,16) by Cyclin-dependent kinase 5 (Cdk5) (17) or c-Jun NH2-terminal kinase (JNK) (18). Interestingly, this phosphorylated form of APP (termed here pAPP) localizes to neurites of cultured hippocampal neurons (17) and is highly concentrated at the terminals of processes in PC12 cells (15). It was proposed that pAPP might play a role in axonal transport (19) and neurite outgrowth during neuronal differentiation (15). However, the fact that APP is phosphorylated in cells other than neurons (20) suggests that phosphorylation of APP may play a more general role. Here we show that a phosphorylated, carboxy-terminal fragment of APP is present in the nucleus of a large variety of cells, including differentiated neurons. We further show that exogenously expressed C{gamma} and Fe65, like the endogenous, phosphorylated APP fragment, accumulate at intranuclear sites that correspond to the splicing factor compartment (SFC), and that Fe65 may regulate this localization. These observations suggest a role for APP in pre-mRNA splicing that may be regulated by APP phosphorylation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antibodies to pAPP detect intracellular pools of phosphorylated APP and C{gamma}
A form of APP, phosphorylated at Thr668, is preferentially localized to neurite endings of cultured neurons (15). To investigate in more detail this localization, we have used a polyclonal antibody raised to a polypeptide from the APP cytoplasmic domain that contained phosphorylated Thr668 (see Materials and Methods). In western blots, this antibody recognized the phosphorylated, but not the non-phosphorylated polypeptide that encompassed the entire cytoplasmic domain of APP (Fig. 1J). In lysates of CAD cells transfected with APP695, the anti-pAPP antibody detected three bands (Fig. 1K) corresponding to immature and mature (partially and fully glycosylated and sulfated) forms of APP695 (see also 21). Consistent with previous reports (15), a higher level of phosphorylation was detected in the mature APP form (upper polypeptide in Fig. 1K). In immunocytochemistry, the anti-pAPP antibody preferentially labeled the neurite terminals of CAD cells (Fig. 1A–C) and of cortical neurons in culture (data not shown) (15,19). In addition, the antibody labeled intranuclear, speckle-like structures (Fig. 1A–G). The labeling was specific, since it was completely eliminated when immunostaining was performed in the presence of competing, phosphorylated (Fig. 1H and I), but not non-phosphorylated (Fig. 1F and G) polypeptide encompassing the APP cytoplasmic domain.



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  		Figure 1. Phosphorylated APP (pAPP) epitopes are localized to the nuclei of differentiated CAD cells. (A–G) An anti-pAPP antibody labels distal neurites (large arrows) and intranuclear, speckled structures (small arrows). Note the necklace-like appearance of the labeled intranuclear structures (A, B, D, F). Labeling is blocked by competition with phosphorylated (H, I), but not non-phosphorylated (F, G) polypeptide encompassing the APP cytoplasmic domain. Corresponding phase contrast micrographs are shown in (C, E, G, I). Scale bars: 50 µm (A), 20 µm (B–I). (J) Western blots of synthetic, phosphorylated (pAPPCD) and non-phosphorylated (APPCD) polypeptides (6 µg/lane), corresponding to the entire APP cytoplasmic domain, probed with antibodies to pAPP (2451; {alpha}-pAPP) or total, phosphorylated and non-phosphorylated, APP (2452; {alpha}-APP). The two antibodies were raised against synthetic, phosphorylated or non-phosphorylated peptides from the cytoplasmic domain of APP (see Materials and Methods). Note that the anti-pAPP antibody does not recognize the non-phosphorylated APP polypeptide, while antibody 2452 ({alpha}-APP) detects both polypeptides. Ponceau S staining of the blot confirms equal load. (K) Western blot of extracts of CAD cells, transfected with non-tagged APP695, probed with antibodies to pAPP and total (i.e. phosphorylated plus non-phosphorylated) APP. Total APP was detected with antibodies to amino-terminal (MAB348; {alpha}-APP-N) and carboxy-terminal (2452; {alpha}-APP) epitopes. Note that the anti-pAPP antibody preferentially detects the fully matured form of APP (arrow). (L) Western blot of a low-speed pellet, prepared from rat brain homogenate, probed with antibodies to amino-terminal ({alpha}-APP-N), carboxy-terminal ({alpha}-APP), and phosphorylated ({alpha}-pAPP) epitopes of APP. The arrow points to low molecular size, proteolytic fragments of APP that are not detected with the antibody to amino-terminal epitopes. No labeling is detected in the blot incubated with anti-pAPP antibody in the presence of competing polypeptide, used at 100 : 1 molar excess over IgG (lane marked with an asterisk). The competing polypeptide was a phosphorylated polypeptide corresponding to the entire cytoplasmic domain of APP. Molecular size markers are in kDa.

 
We reasoned that the intranuclear labeling was likely due to phosphorylated C{gamma} (pC{gamma}), an APP fragment previously shown to translocate into the nucleus (22). Indeed, tagged C{gamma} accumulated in the nuclei when expressed at moderate levels in CAD cells (Fig. 2A–D). Like the endogenous anti-pAPP immunoreactivity, exogenously expressed C{gamma} localized to intranuclear speckles (Fig. 2A and C). At high levels of expression, C{gamma} was detected both in nuclei and cytoplasm, throughout the cell (Fig. 2E). Compared with non-transfected cells, labeling with the anti-pAPP antibody was highly increased in CAD cells transfected with C{gamma} (Fig. 2E–K). This result suggested that, in addition to endogenous pAPP epitopes, the anti-pAPP antibody detected the fraction of exogenously expressed C{gamma} that becomes phosphorylated. Indeed, the labeling with the anti-pAPP antibody fully co-localized with C{gamma}-green fluorescent protein (GFP) (Fig. 2E–K) or C{gamma}-myc (data not shown) in the cytoplasm (Fig. 2E–H) and, most importantly, at intranuclear speckles (Fig. 2I–K). Competition with phosphorylated, but not non-phosphorylated APP polypeptide eliminated all labeling, confirming specificity of staining for pAPP epitopes (data not shown). In a separate set of experiments, we tested whether C{gamma}, generated from exogenously expressed, full length APP through the action of endogenous secretases, also becomes localized to intranuclear speckles. To this end, CAD cells were transfected with an APP–yellow fluorescent protein (YFP) construct (YFP being fused to the carboxy-terminus of human APP695) (23), and processed for direct visualization of YFP fluorescence and immunocytochemical detection of pAPP epitopes. As expected, most APP-YFP was detected in the cytoplasm. However, in cells that expressed high levels of APP-YFP, a small amount of the YFP tag was occasionally detected in the nucleus, where it co-localized with pAPP epitopes at speckles (Fig. 3A–F). These experiments indicated that a fragment containing the YFP-tagged, carboxy-terminal region of APP-YFP (probably C{gamma}-YFP; Fig. 3G) was targeted to intranuclear speckles. Taken together, these results indicate that: (1) C{gamma} is phosphorylated in neurons; (2) pC{gamma} can be detected with the anti-pAPP antibody in immunocytochemistry; and (3) pC{gamma} localizes to intranuclear speckles.



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  		Figure 2. Exogenously expressed C{gamma} localizes to intranuclear speckles. (A–D) Localization of GFP-tagged C{gamma} (C{gamma}-GFP) to necklace-like, intranuclear structures in CAD cells. (E–K) C{gamma}-GFP, visualized by GFP fluorescence, co-localizes with epitopes detected with the anti-pAPP antibody in the cytoplasm (E–H) and at intranuclear speckles (I–K). Non-transfected cells (arrows) show intranuclear anti-pAPP immunoreactivity exclusively at speckles (F, G, J, K). Corresponding phase contrast micrographs are shown in (B, D, H). Scale bars: 20 µm. (L) Western blots of extracts of non-transfected CAD cells (control) and of CAD cells transfected with C{gamma}-GFP probed with antibodies to total (2452; {alpha}-APP) or phosphorylated ({alpha}-pAPP) APP and to GFP ({alpha}-GFP). Note that a polypeptide of ~7 kDa, likely to be endogenous C{gamma}, is detected with a longer exposure in transfected cells (bottom blots, arrow). Molecular size markers are in kDa.

 


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  		Figure 3. The intracellular domain of exogenously expressed APP-YFP localizes to intranuclear speckles. (A–F) The YFP-tag co-localizes with epitopes detected with anti-pAPP antibody in the nuclei of CAD cells transfected with APP695-YFP (YFP is fused to the carboxy-terminus of APP). Arrows in (A–C) point to a transfected cell. Corresponding phase contrast micrographs are shown in (C, F). Scale bars: 20 µm. (G) A phosphorylated, YFP-tagged APP carboxy-terminal fragment is contained in the nuclear fraction. CAD cells, transfected with APP695–YFP, were subfractionated into a nuclear pellet (nuclei) and a postnuclear supernatant (supe; 10% load relative to nuclei), and analyzed by western blotting with anti-pAPP ({alpha}-pAPP) and anti-YFP ({alpha}-YFP) antibodies. A polypeptide corresponding by size to C{gamma}-YFP (big arrow) is detected in the nuclear fraction. At longer exposure, a polypeptide of ~7 kDa, likely to be endogenous C{gamma}, is also detected with the anti-pAPP antibody (small blot at left, arrow). Full-length, phosphorylated APP-YFP and endogenous pAPP are largely detected in the membrane-rich postnuclear supernatant. Small amounts of full-length pAPP and pAPP-YFP are also found in the nuclear fraction (bracket), probably due to contamination with endoplasmic reticulum (22).

 
To confirm biochemically that a phosphorylated APP polypeptide that might correspond to C{gamma} is enriched in the nucleus, immunoblots were performed of total and nuclear extracts prepared from rat brain, non-transfected CAD cells, and CAD cells transfected with C{gamma}-GFP or full-length APP-YFP. In western blots of rat brain preparations enriched in nuclei, the anti-pAPP antibody detected small polypeptides in a size range that includes C{gamma} (~7 kDa; Fig. 1L). It was not possible to detect by western blotting endogenous pC{gamma} neither in the cell lysate nor in the nuclear fraction prepared from non-transfected CAD cells. This result was expected, since in normal conditions C{gamma} is undetectable, due to its rapid degradation (12,22). However, the anti-pAPP antibody recognized a ~7 kDa polypeptide in homogenates and nuclear preparations obtained from CAD cells transfected with either C{gamma}-GFP (Fig. 2L) or full length APP-YFP (Fig. 3G). This polypeptide probably represents the endogenous pC{gamma}, since it was only detected with antibodies to the cytoplasmic domain of APP, but not with antibodies to epitopes in the Aß region or in the extracellular domain of APP (Fig. 2L and data not shown). The anti-pAPP immunoblots also showed polypeptides that migrate at positions corresponding to C{gamma}-GFP (Fig. 2L) and C{gamma}-YFP (Fig. 3G), the latter being generated by intracellular processing from transfected APP-YFP. As expected, these polypeptides were also recognized by an antibody to GFP (Figs 2L and 3G), and were absent in preparations from non-transfected cells (Fig. 2L). Figure 3G also shows that the phosphorylated APP polypeptide corresponding to pC{gamma}-YFP was only detected in the nuclear fraction, and not in the postnuclear supernatant prepared from CAD cells transfected with APP-YFP. As described above, while endogenous pC{gamma} is not detected in western blots of non-transfected CAD cells, it becomes detectable in cells transfected with tagged C{gamma} or APP. This is probably due to either a decrease in overall degradation of C{gamma} (as a consequence of increased substrate amount) or some other mechanism of stabilization of endogenous C{gamma} in the presence of increased concentration of exogenous C{gamma}. Some of this non-tagged pC{gamma} may also result from proteolysis of tagged pC{gamma}. Together with the data of immunolocalization, these results indicate that pC{gamma} is enriched in the nuclei of CAD cells. Further studies are required to determine whether nuclei also contain phosphorylated APP polypeptides other than pC{gamma}.

pAPP-positive nuclear bodies co-localize with the splicing factor compartment
The intranuclear distribution of endogenous pAPP epitopes (Fig. 1) and of exogenously expressed C{gamma} (Fig. 2) was reminiscent of the nuclear speckles that define the SFC (24). The subnuclear structures labeled by the anti-pAPP antibody had either a necklace appearance (Fig. 1) or consisted of a few, large dots. Occasionally, a more diffuse, but still dotted labeling pattern was observed. Double labeling immunocytochemistry was used to confirm co-localization of anti-pAPP immunoreactivity with the SFC. As shown in Figure 4, anti-pAPP immunostaining co-localized to a large extent with bona fide residents of the SFC (25): the small nuclear ribonucleoprotein (snRNP), U2B (Fig. 4A–D) and the serine/arginine-rich (SR) proteins (Fig. 4E–H). No co-localization was seen with survival of motor neuron (SMN) protein (Fig. 4I–L), a resident, for the most part, of other intranuclear structures, the coiled bodies and the gems (26–28).



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  		Figure 4. Intranuclear, anti-pAPP immunoreactivity co-localizes with the splicing factor compartment. Note that pAPP epitopes co-localize with the snRNP, U2B (A–D, M–O) and SR proteins (labeled with mAb104; E–H), but not with the SMN protein (I–L, P–R) in CAD cells (A–L) and cortical neurons (M–R). Corresponding phase contrast micrographs are shown in (D, H, L). Scale bars: 10 µm (A–L), 20 µm (M–R).

 
In cortical neurons from mouse brain (Figs 4 and 5), the anti-pAPP antibody labeled intranuclear structures that appear similar to those described in CAD cells. As in CAD cells, anti-pAPP staining co-localized with U2B protein (Fig. 4M–O) and SR proteins (data not shown), but not with the SMN protein (Fig. 4P–R). This confirms localization of pAPP epitopes to the SFC in freshly isolated neurons that most likely reflect the physiological situation. Since APP is ubiquitously expressed, we asked whether pAPP epitopes are present in the nuclei of cells other than neurons. As shown in Figure 5, intranuclear labeling was detected in all cell types tested: COS-1 (Fig. 5C and D), MDCK (Fig. 5E and F), bovine aortic endothelial cells (Fig. 5G and H), and human microvascular endothelial cells (data not shown). It is concluded that pC{gamma} accumulates intranuclearly in many cell types, and is targeted to the SFC.



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  		Figure 5. The anti-pAPP antibody labels intranuclear speckles in mouse cortical neurons (MCN; A, B), COS-1 (C, D), MDCK (E, F), and bovine aortic endothelial (BAEC; G, H) cells. Corresponding phase contrast micrographs are shown in (B, D, F, H). Scale bars: 10 µm (A, B), 20 µm (C–H).

 
The scaffolding protein, Fe65 co-localizes with C{gamma} at the splicing factor compartment
The scaffolding protein, Fe65 has been implicated in the stabilization and nuclear translocation of C{gamma}. Indeed, previous studies using overexpressed proteins indicated that C{gamma} interacts with Fe65 both in the cytoplasm and in the nucleus (11–13). Here, this observation has been extended by showing that, in the nucleus, exogenously expressed C{gamma} and Fe65 co-localize at sites that correspond to the SFC (Fig. 6A–C). This suggests not only that C{gamma} and Fe65 may be transported to the SFC as a complex, but also that they may retain their association at this destination. We also noticed that the intensity of the intranuclear labeling with the anti-pAPP antibody is sensitive to the intracellular concentration of Fe65. Thus, in CAD cells that overexpressed myc-tagged Fe65 at relatively low levels, immunolabeling with the anti-pAPP antibody co-localized with Fe65 at the SFC (Fig. 6D–F). At high levels of expression of Fe65-myc, less anti-pAPP immunoreactivity was detected in the nucleus, compared with cells that did not express exogenous Fe65 (Fig. 6G, H and I). This result indicates that Fe65 may regulate, directly or indirectly, intracellular processes that lead to the localization of pAPP epitopes to the SFC.



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  		Figure 6. Exogenously expressed Fe65 co-localizes with transfected C{gamma} (A–C) and with endogenous pAPP epitopes (D–F) at intranuclear speckles. CAD cells were transfected with Fe65-myc alone (D–H) or co-transfected with Fe65-myc and C{gamma}-GFP (A–C). Note that anti-pAPP immunoreactivity is diminished in cells that express Fe65 at high level (arrows, G, H). Scale bar: 20 µm. (I) Quantitative measurement of the effect of overexpression of JIP1–FLAG and Fe65–myc on intranuclear anti-pAPP immunoreactivity. The graph shows percentages of transfected CAD cells that showed anti-pAPP labeling at levels comparable or higher than in surrounding, non-transfected cells.

 
Phosphorylated APP epitopes redistribute during mitosis
During mitosis, components of the SFC, such as the SR proteins, redistribute throughout the cytoplasm, in a particulate fashion (29,30). As shown in Figure 7, pAPP immunoreactivity in mitotic CAD (Fig. 7A–C) and MDCK (Fig. 7D–F) cells also displayed a particulate pattern. This result suggests that pAPP polypeptides remain associated with components of the SFC throughout the cell cycle. However, compared with cells in interphase, the level of anti-pAPP immunoreactivity detected in mitotic cells was largely increased, an indication that additional phosphorylation of APP, possibly unrelated to the dispersion of speckles, occurs at mitosis. This result is consistent with data of biochemistry showing enhanced phosphorylation of APP during the G2/M phase of the cell cycle (20).



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  		Figure 7. APP is hyperphosphorylated, and distributes in a particulate fashion, throughout the cytoplasm of mitotic CAD (A–C) and MDCK (D–F) cells. Cells were stained with the anti-pAPP antibody (shown in A, B, D, F). The mitotic spindle, labeled with anti-tubulin antibody, is shown in E (Tub). (A and B) show the same field; contrast is enhanced in (B) to reveal intranuclear labeling in non-mitotic cells. A corresponding phase contrast micrograph is shown in (C). Scale bars: 20 µm (A–C), 10 µm (D–F).

 
It was proposed that phosphorylation of APP at the G2/M phase of the cell cycle is performed by Cdc2 (20). In neurons, JNK and Cdk5 are candidate kinases for APP phosphorylation (17,18). In an attempt to identify the kinase that is responsible for phosphorylation of intranuclear APP polypeptides, we tested the effect of blocking the activities of JNK and Cdk5 on the anti-pAPP immunoreactivity. As shown in Figure 8A and B, anti-pAPP immunoreactivity was detected in cells treated with the Cdk5 inhibitor, roscovitine. Similarly, expression of a dominant negative Cdk5 (dnk5-GFP) (31) did not diminish intranuclear anti-pAPP immunoreactivity (data not shown). To block JNK activity, dominant negative strategy was used that employs transfection of cells with the JNK interacting protein 1 (JIP1), a scaffolding protein for JNK (32). Since overexpression of JIP1 causes marked inhibition of JNK by sequestering it in the cytoplasm, away from its substrates, this experimental condition is considered to be the most specific inhibitor for JNK (33,34). JIP1 overexpression in CAD cells considerably reduced phosphorylation of cytoplasmic APP (manuscript submitted). However, no effect on the level of intranuclear anti-pAPP immunoreactivity was seen in cells that overexpressed JIP1-FLAG (Fig. 8C–E). It is concluded that cytoplasmic APP and intranuclear APP polypeptides are phosphorylated by different kinases.



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  		Figure 8. Inhibition of Cdk5 and JNK activity does not block phosphorylation of intranuclear APP polypeptides. Note localization of anti-pAPP immunoreactivity to intranuclear speckles in CAD cells treated with the Cdk5 inhibitor, roscovitine (A, B) and in a cell expressing JIP1–FLAG (arrow in C–E). JIP1 is detected with an anti-FLAG antibody (D). Corresponding phase contrast micrographs are shown in (B, E). Scale bar: 20 µm.

 
Antibodies to the carboxy-terminus of APP label intranuclear speckles
To further characterize the nature of the speckle-associated pAPP antigen, CAD cells were immunolabeled with antibodies raised to distinct epitopes from the APP cytoplasmic domain. First, a polyclonal antibody (AB5352; Chemicon) was used, raised to a nine amino acid polypeptide from the carboxy-terminus of APP. This antibody should recognize full-length APP as well as carboxy-terminal fragments, including C{gamma}, independent of their state of phosphorylation (35,36). When used in immunocytochemistry, this antibody labeled the cell body and processes of CAD cells, although with low sensitivity (Fig. 9A and B). Upon careful examination of the cell body labeling, we noticed that it was mostly confined to the nuclei, where it clearly distributed in a speckled fashion, much like the labeling pattern detected with the anti-pAPP antibody (Fig. 9C–G). Next, a polyclonal antibody (2452; Cell Signaling Technology) was used, raised to a non-phosphorylated, synthetic polypeptide that encompassed residues surrounding Thr668 of APP695. This antibody detects full-length APP and carboxy-terminal cleavage products of APP. In addition to non-phosphorylated APP, this antibody also detects phosphorylated APP (Fig. 1J). In CAD cells (Fig. 9H–M) and primary cortical neurons (data not shown), the antibody heavily labeled the endoplasmic reticulum-Golgi region, and less intensely the processes. In both cell types, the antibody also labeled intranuclear structures, which often had necklace-like appearance (Fig. 9J and data not shown).



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  		Figure 9. Antibodies to the carboxy-terminal region of APP label intranuclear speckles. CAD cells were processed for immunocytochemistry with antibody AB5352, raised to a peptide corresponding to the nine carboxy-terminal amino acids of APP (A–G), with antibody 2452, raised to a synthetic polypeptide corresponding to residues surrounding Thr668 of APP695 (H–M), or with antibody 4G8, reactive to an epitope in the Aß region of APP (N, O). These antibodies detect APP independent of its phosphorylation state. (A and B) show the same field; contrast is enhanced in (B) to reveal intranuclear labeling. (C–G) Gallery of representative images, showing the speckled appearance of intranuclear labeling. Note that the antibody 4G8, reactive to an Aß epitope, does not label nuclei (compare N with H, L). Arrows in (A, B, H) point to cells shown at higher magnification in (C, D, J). Corresponding phase contrast micrographs are shown in (I, K, M, O). Scale bars: 50 µm (A, B, H, I, L–O); 20 µm (C–G, J, K). (P) Schematic diagram of APP695 showing the position of peptides (b, d–i) that served as antigens to generate some of the anti-APP antibodies used in immunocytochemistry. The position of the epitopes recognized by antibody MAB348 (Chemicon; a) and 4G8 (Signet; c) are also shown. The Aß region (gray), the YENPTY motif (marked with Y), and Thr668 (T) are indicated. The peptides correspond to the following antibodies: b, anti-rodent Aß (Signet); d, anti-APP (2452; Cell Signaling); e, anti-pAPP (2451; Cell Signaling; pT denotes that the peptide contains phosphorylated threonine); f, antibody G369 (developed by S. Gandi; 42,43); g, antibody AB5352; (Chemicon); h, antibodies C7, C8 (12,13,68), and anti-APP, C-terminal (Sigma) (38); i, anti-APP, CT695 (Zymed) (39).

 
Antibodies raised to epitopes surrounding the YENPTY motif in the carboxy-terminal region of APP, largely used in immunocytochemical detection of APP, were less efficient in labeling intranuclear structures (data not shown). This is probably explained by the fact that this region of APP is blocked in situ by the interaction with APP binding proteins, including Fe65 (4), and is thus inaccessible to the antibody. We also tested antibodies raised to epitopes outside the C{gamma} region of APP: MAB348 (Chemicon International; recognizing an amino-terminal epitope), anti-rodent Aß (Signet Laboratories Inc.), and 4G8 (Signet; recognizing an epitope from the Aß region of APP). None of these antibodies labeled intranuclear structures, even in cells that had been transfected with APP695 (Fig. 9N and O and data not shown). Taken together, these results of immunocytochemistry confirm the presence at nuclear speckles of polypeptides from the APP cytoplasmic domain, such as C{gamma}. Future studies, using antibodies that detect only non-phosphorylated APP species (currently under development), are required to determine whether intranuclear speckles also contain non-phosphorylated C{gamma} in addition to pC{gamma}.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To understand the role of phosphorylation in APP transport and metabolism, the localization of Thr668-phosphorylated forms of APP in cultured neurons, epithelial cells and fibroblasts has been investigated. With the aid of an antibody that specifically detects phosphorylated, but not non-phosphorylated forms of APP (full length and carboxy-terminal fragments) a previously unknown localization of the phosphorylated cytoplasmic fragment of APP has been identified: the intranuclear SFC. This localization is probably a general feature of APP, since it was detected in neuronal and many non-neuronal cells. The identity of the nuclear speckles, where pAPP epitopes accumulate, was established by co-localization with SR splicing factors and the snRNP, U2B, which are enriched in the SFC (24,25). The localization of the APP carboxy-terminal fragment to the SFC was confirmed with two additional antibodies raised to polypeptides from the APP cytoplasmic domain.

A large body of data indicates that the intranuclear molecular species detected with the anti-pAPP antibody represents a bonafide, phosphorylated APP fragment from its carboxy-terminal end, probably pC{gamma}. First, antibodies raised to a different epitope in the APP cytoplasmic domain (contained in C{gamma}) reveal a similar labeling pattern. Second, the staining with the anti-pAPP antibody co-localizes with exogenously expressed C{gamma}, as detected by its tag. Third, intranuclear anti-pAPP immunoreactive sites co-localize with YFP-labeled, carboxy-terminal fragments generated from exogenously expressed APP-YFP. Fourth, pC{gamma}–YFP and endogenous pC{gamma} are biochemically detected in a nuclear fraction prepared from APP-YFP expressing cells. In addition, the intranuclear anti-pAPP immunoreactivity co-localizes with Fe65, a scaffolding protein known to be associated with C{gamma} in the nucleus (13).

Consistent with the results reported here, numerous studies have found that exogenously expressed C{gamma} accumulates in the nucleus (12,14,37–39), with or without co-expression of Fe65. Moreover, in line with our results, an APP carboxy-terminal fragment corresponding to C{gamma} was detected biochemically in cells that overexpressed full-length APP (13). Although these studies did not investigate in detail the intranuclear localization of C{gamma}, they do occasionally show images suggesting a speckled distribution of this APP polypeptide in the nucleus (37).

Using antibodies raised to polypeptides from the carboxy-terminal region of APP, previous studies did not detect APP polypeptides at the SFC (12,13,38,39). However, the antibodies used in those studies were raised to domains surrounding the YENPTY motif, likely to be blocked by interactions with proteins that contain a phosphotyrosine-binding (PTB) domain, such as Fe65 (40) (Fig. 9P). By contrast, the antibodies employed in this study were raised to polypeptides that correspond to regions in the APP cytoplasmic domain that are unlikely to interact in the nucleus with any of the known APP binding proteins. Indeed, the region surrounding Thr668 (used as antigen for the anti-pAPP and anti-APP antibodies from Cell Signaling Technology) has been reported to interact only with the Go heterotrimeric GTPase, unlikely to be present in the nucleus (4,41). Also, no protein has been shown to bind to the region encompassing the nine carboxy-terminal amino acids of APP (used as antigen for the generation of the antibody AB5352 from Chemicon International; see Fig. 9P). Note that, in an earlier report (42), a polyclonal antibody, raised against a polypeptide corresponding to an extended carboxy-terminal region of APP695 that included Thr668 (amino acids 645–694) (43) readily detected endogenous C{gamma} in the nuclei of brain neurons.

What could be the function of C{gamma} at the SFC? This nuclear subcompartment is enriched in splicing factors (mostly SR proteins), transcription factors, snRNAs, ribosomal proteins, kinases and phosphatases (24,25). It is thought that the speckles serve as storage and assembly sites for the splicing machinery, regulating concentration and availability of splicing factors, and thus regulating pre-mRNA splicing. C{gamma} may be part of this machinery and could play a role in modulating pre-mRNA splicing. Interestingly, the association of splicing factors with speckles appears to be regulated by phosphorylation. Both the SR proteins and the snRNPs are subjected to reversible phosphorylation, which causes them to be released from, or recruited to the SFC (44). Similarly, phosphorylation of C{gamma} may regulate its transport to, and association with the speckles. Rather than being released from the speckles, as phosphorylated SR proteins are (44), pC{gamma} appears to be recruited to the speckles.

The localization of C{gamma} at the SFC is also consistent with its proposed role in regulating transcription (11,14,45). Numerous lines of evidence suggest that transcription and splicing are physically and functionally coupled, and many transcription factors become localized to the SFC (25). Moreover, both splicing and transcription generally occur at the periphery of the SFC, where transcription factors and acetylated histones become localized during active gene transcription (46). Importantly, the histone acetyltransferase, Tip60, proposed to form a complex with Fe65 and C{gamma} in the nucleus (11) localizes to intranuclear speckles (47–49) that may correspond to the SFC. In addition, the proteolytically cleaved, intracellular domain of LRP, proposed to modulate APP signaling, also localizes to intranuclear speckles (47). Further studies are needed to address whether C{gamma} participates in the regulation of transcription, pre-mRNA splicing, or in both processes.

Another novel finding of this study is that a substantial fraction of exogenously expressed C{gamma} is phosphorylated at the threonine corresponding to Thr668 of APP695. In differentiated neurons, only a small fraction of full-length APP is phosphorylated at this residue, and becomes localized at neurite terminals (15). In this context, it was proposed that APP phosphorylation might play a role in neuronal differentiation and vesicular transport of APP into neurites (15,19). Our study shows that C{gamma} can be efficiently phosphorylated as a soluble polypeptide, when expressed in CAD cells. This raises the question whether phosphorylation of endogenous, intranuclearly localized C{gamma} occurs prior to, or after its release from APP by the action of {gamma}-secretase. With regard to its possible function, phosphorylation of C{gamma} may either extend its half-life, or regulate its interaction with other proteins or RNAs. C{gamma} has been reported to interact with several proteins (4), and phosphorylation may modulate these interactions (50). While C{gamma} is not known to interact with nucleic acids, it is interesting to note that the amyloid precursor-like protein, APLP2, was initially identified as the DNA binding protein, CDEI-binding protein (CDEBP) (51). Recent studies have shown that amyloid precursor-like proteins can undergo, like APP, regulated intramembrane proteolysis, a process that releases C{gamma}-like intracellular domains (52). Since phosphorylation of APLP2 at a threonine that corresponds to Thr668 in APP has been reported (53), some of the endogenous immunoreactive species detected in the nucleus with the anti-pAPP antibody may represent the intracellular fragment released from APLP2 rather than that of APP. The contribution of one or the other intra-cellular fragment remains to be determined in future studies.

What kinase phosphorylates intranuclearly localized C{gamma}? Previous studies have shown that Cdc2 phosphorylates full-length APP in non-differentiating cells during the G2/M phase of the cell cycle (20). In neurons, both Cdk5 and JNK have been proposed to phosphorylate APP (17,18). Using a dominant negative strategy to block JNK activity in CAD cells, it has been shown that this kinase probably phosphorylates cytoplasmic APP (Z. Muresan and V. Muresan, submitted for publication). A limited investigation using dominant negative approaches and specific inhibitors indicated that neither Cdk5 nor JNK phosphorylates intranuclearly localized C{gamma}. This suggests that phosphorylation of intranuclear C{gamma} may occur by a different mechanism than phosphorylation of cytoplasmic APP. Since pC{gamma} is present in the nuclei of a large variety of cells, both differentiated and non-differentiated, it is likely that its phosphorylation is performed by a kinase of ubiquitous distribution.

What mechanism targets C{gamma} to the SFC? It has been suggested that the scaffolding protein, Fe65, stabilizes C{gamma} and facilitates its translocation into the nucleus (11–13). It was found that, when expressed at low levels in CAD cells, Fe65 co-localizes with endogenous pAPP epitopes and with exogenously expressed C{gamma} at intranuclear speckles. Moreover, high levels of expression of Fe65 appeared to decrease the amount of endogenous pAPP epitopes associated with speckles by a mechanism that remains to be identified. Taken together, these results suggest that Fe65 and C{gamma} are closely associated at the SFC, but do not rule out independent transport of the two proteins into the nucleus (37). Our study did not address the possibility that C{gamma} associates with JIP1 in the nucleus, as recently reported (39). Nevertheless, our unpublished results (manuscript submitted) indicate that pC{gamma} preferentially interacts with JIP1 in vitro, suggesting that such an interaction might also occur in vivo.

While this and other studies (11,45) point to a role of APP and Fe65 in regulating intranuclear processes, the two proteins appear to also function together outside the nucleus. Thus, it has been proposed that an APP–Fe65 complex participates in the regulation of actin-based membrane motility in neurons, by localizing to growth cones and synapses (54,55). APP may not be unique in having separate functions in the nucleus and at the synapse. The SMN protein, which is deleted or mutated in spinal muscular atrophy (56), is localized to both the nucleus and growth cones of neurons and glial cells (57,58). It has been proposed that SMN functions in spliceosome assembly and pre-mRNA splicing (59,60). Outside the nucleus, SMN may play a role in neurite outgrowth and neuromuscular maturation (57,58). Recent data indicate that only a truncated isoform of SMN, lacking exon-7, accumulates into the nucleus (58). In the case of APP, nuclear localization requires proteolytic processing of the protein, to generate C{gamma}. Further studies are required to investigate the function of the APP fragments in the nucleus, including the possible role of C{gamma} in pre-mRNA splicing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Antibodies
Primary antibodies used in this study were: rabbit anti-APP (2452, raised against a KLH coupled synthetic peptide corresponding to residues surrounding Thr668 of human APP695; detects APP independent of its phosphorylation state; Cell Signaling Technology, Beverly, MA, USA); rabbit anti-phospho-APP (Thr668) (2451, raised against a KLH coupled synthetic phosphopeptide corresponding to residues surrounding Thr668 of human APP695; does not cross-react with non-phosphorylated APP; Cell Signaling Technology); rabbit anti-APP, carboxy-terminal (AB5352, raised to a nine amino acid peptide from the carboxy-terminus of APP; Chemicon International, Temecula, CA, USA); rabbit anti-rodent Aß (Signet Laboratories Inc., Dedham, MA, USA); mouse anti-human Aß (4G8, Signet; reacts with an epitope conserved in rodents); and mouse anti-APP (MAB348, clone 22C11, recognizing an amino-terminal epitope in APP; Chemicon International). The monoclonal antibody 4G3 recognizing the snRNP, U2B (61) was a gift from Dr Helen Salz (Case Western Reserve University, Cleveland, OH, USA). The pan-SR proteins monoclonal antibody, mAb104 (62) was a gift from Dr Jim Bruzik (Case Western Reserve University, Cleveland, OH, USA). A monoclonal antibody to the SMN protein (BD Biosciences Pharmingen, San Diego, CA, USA) was a gift from Dr Greg Matera (Case Western Reserve University, Cleveland, OH, USA). A monoclonal antibody to {alpha}-tubulin was from Sigma (St Louis, MO, USA); a monoclonal antibody to GFP (JL-8, BD Living ColorsTM; cross-reacts with YFP) was from BD Biosciences Clontech (Palo Alto, CA, USA); a monoclonal anti-c-myc antibody (Ab-1; Clone 9E10) was from Oncogene Research Products (San Diego, CA, USA); and a monoclonal anti-FLAG antibody (M2) was from Stratagene (La Jolla, CA, USA).

Cell culture
The mouse central nervous system, catecholaminergic cell line, CAD (63), was grown in 1 : 1 F12 : DME medium, containing 8% fetal bovine serum (FBS) and penicillin–streptomycin. CAD cell differentiation was induced by serum removal from the culture medium, as described (63). E16.5 day mouse cortical neurons were grown in Neurobasal Medium with B-27 supplement, L-glutamine, and penicillin–streptomycin for 5 days, with a change of media on day 4. Neurons were maintained in growth media until they were fixed for immunocytochemistry. In some experiments, CAD cells were treated with the potent Cdk5 inhibitor, roscovitine (Calbiochem, San Diego, CA, USA; 20 µM, overnight) (31).

MDCK and COS-1 cells were grown in high glucose DME medium, containing 10% FBS and penicillin–streptomycin. Bovine aortic endothelial cells (passage 4–10) were cultured in 1 : 1 F12 : DME medium, supplemented with 5% FBS and penicillin–streptomycin. Human microvascular endothelial cells were cultured in EGM-2 (Cambrex, Walkersville, MD, USA).

Transfection
CAD cells were transfected according to the manufacturer's instructions, by using FuGene 6 (Roche Diagnostics, Indianapolis, IN, USA). EGFP- and myc-tagged carboxy-terminal fragment of APP (C58-GFP and C58-myc; called throughout the paper C{gamma}-GFP and C{gamma}-myc) (13) were gifts from Dr Ayae Kinoshita and Dr Bradley Hyman (Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA). Myc-tagged Fe65 (cloned into RK5 vector) (10) was a gift from Dr Ben Margolis (University of Michigan, Howard Hughes Medical Institute, Ann Arbor, MI, USA). YFP-tagged human APP695 (23) was a gift from Dr Christoph Kaether (European Molecular Biology Laboratory, Heidelberg, Germany). The pcMV–FLAG–JIP1 cDNA (32) was a gift from Dr Roger Davis (University of Massachusetts Medical School, Howard Hughes Medical Institute, Worcester, MA, USA). The dnk5–GFP cDNA, encoding a dominant negative form of Cdk5 (31) was a gift from Dr Li-Huei Tsai (Harvard Medical School, Howard Hughes Medical Institute, Boston, MA, USA).

Immunocytochemistry
Transfected or non-transfected CAD, COS-1, MDCK, and endothelial cells, and primary cultures of cortical neurons were fixed for 20 min in PBS containing 4% formaldehyde and 4% sucrose, then permeabilized with 0.3% Triton X-100 (20 min at 20°C), and processed for single or double antigen labeling as previously described (64,65). Secondary antibodies coupled to Alexa dyes were from Molecular Probes (Eugene, OR, USA). In control experiments, immunolabeling with anti-pAPP antibody was done in the presence of phosphorylated or non-phosphorylated polypeptides (100 : 1 molar excess over IgG) that encompassed the entire APP cytoplasmic domain. Digital images were obtained with a Nikon Optiphot microscope (100x oil, 40x, and 20x objectives) equipped with a cooled CCD camera, and collected using Optronics Magnafire image analysis software. In some cases, samples were analyzed by confocal microscopy.

Other procedures
Biotinylated polypeptides corresponding to the entire cytoplasmic domain of APP were synthesized with either phosphorylated or non-phosphorylated threonine (corresponding to Thr668 in APP695). Preparation of rat brain (P10) and CAD cell homogenates, and subcellular fractionation were done as previously described (66). A low-speed pellet that contained nuclei, large membrane compartments and cytoskeletal elements was prepared from rat brain homogenate by centrifugation (10 min at 1000g). Nuclear fractions and postnuclear supernatants were prepared from transfected CAD cells according to published procedures (12,67). Briefly, cells were washed in PBS, incubated for 10 min on ice in hypotonic buffer (10 mM Tris–HCl, pH 7.4, containing 10 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and a mixture of protease inhibitors), and Dounce homogenized. The homogenate was separated into a nuclear fraction and a postnuclear supernatant by 5 min centrifugation at 400g. Electrophoresis (10% SDS–PAGE for separation of rat brain proteins; 12% SDS–PAGE for separation of CAD cell extracts; 16.5% Tris–Tricine for polypeptide separation) and immunobloting were done as previously described (65).


   ACKNOWLEDGEMENTS
 
We thank Samantha Cicero and Dr Karl Herrup for providing the cortical neuron cultures, and Dr Dona Chikaraishi and Dr James Wang for providing the CAD cell line. We also thank Dr Jim Bruzik, Dr Roger Davis, Dr Carlos Dotti, Dr Brad Hyman, Dr Christoph Kaether, Dr Ayae Kinoshita, Dr Ben Margolis, Dr Greg Matera, Dr Helen Salz, and Dr Li-Huei Tsai for their generous gifts of reagents. We are thankful to Dr Greg Matera and Dr Jim Bruzik for helpful advice. This work was supported by start-up funds from the Department of Physiology and Biophysics, Case Western Reserve University, a Mt Sinai Health Care Foundation Scholarship, and Pilot Grant AG08012 from the Alzheimer's Disease Research Center at the University Hospitals of Cleveland and Case Western Reserve University (V.M.).


   FOOTNOTES
 
* To whom correspondence should be addressed at: Department of Physiology and Biophysics, School of Medicine, E553, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4970, USA. Tel: +1 2163684766; Fax: +1 2163683952; Email: virgil.muresan{at}case.edu


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