Differences In Phosphorylation Levels Research Articles

ODP368 Hyper-phosphorylation of β-catenin at Serine552: predictive marker of invasion and recurrence of Non-Functioning Pituitary Tumours (NFPTs)

Abstract Background NFPTs are the most common operated pituitary tumours and can present with visual field defects, hormone deficiencies and headache. Surgery is treatment of choice but recurrence rate is high ranging from 10-50%, depending on the extent of tumour removal. No confirmed predictive biomarkers for NFPT recurrence have been identified, apart from Ki-67. We applied high-throughput mass spectrometry-based phosphoproteomic approach to explore the phosphorylation pattern of proteins in NFPTs in order to identify predictive markers of invasion and recurrence. Methods Based on radiological, histopathological, and surgical features, NFPTs were sub-grouped into three groups: non-invasive (n=5), invasive (n=10) and recurrent (n=5) subtypes. Invasiveness was determined by radiology (Knosp classification 3&4), histopathological invasion (bone, dura and mucosa) and intraoperative findings. Tumour recurrence was based on radiological data for a mean±SD follow-up of112±39 months. Fresh-frozen pituitary tumour tissues were used for protein extraction and phosphopeptides were enriched using TiO 2 and labelled with tandem mass tags and subjected tomass spectrometry (Orbitrap)for quantification. Candidate hyper-phosphorylated proteins were validated by immunohistochemistry in 200 additional tumour samples by immunoblotting (n=36). Results In total, we identified 3185 phosphopeptides and observed significant difference in phosphorylation levels of invasive and recurrent groups. Compared to non-invasive cases, in invasive group we found, 452 hyper and 93 hypo phosphorylated proteins, while in the recurrent group there were 790 hyper and 307 hypo phosphorylated proteins. Phospho-serine showed the highest level of difference (90.3%) among the groups, followed by threonine (8.9%) and tyrosine (0.8%). One of the top differentially phosphorylated proteins was Ser552 of β-catenin showing significant hyper-phosphorylation in recurrent (p<0. 001) and invasive (p<0. 001) NFPTs. We also observed hyper-phosphorylation in tumours with suprasellar (p<0. 05) and cavernous sinus extension (p<0. 01). There was no correlation with tumour diameter and volume. Receiver operating characteristics curve analysis was performed to find the optimal cut-off value of β-catenin pSer552 immunohistochemical H-score in patients who had recurrence (n=44) or non-recurrence (n=156)and observed an area under curve of 0.717 (95% CI: 0.61-0.80),indicating a good prognostic ability for theβ-catenin pSer552H-score. A cut-off value of 160 for theβ-catenin pSer552H-score gives a sensitivity of 69% and a specificity of 73% for tumour recurrence. Kaplan-Meier survival curve analysis shows strong statistical correlation in the recurrence free survival (p<0. 0001) and the nuclear positive staining of β-catenin pSer552 with a hazard ratio of 3.1 (95% CI 1.5-6.3). Conclusions our study has identified hyper-phosphorylation of β-catenin at the Ser552 in recurrent and invasive NFPT subgroups. The H-score of β-catenin p552 correlates with tumour recurrence free survival in a large cohort of NFPT patients, which supports that β-catenin pSer552 could be used as predictive biomarker for NFPT recurrence. Presentation: No date and time listed

Abstract 3398: PTPN22 is a systemic target for augmenting antitumor immunity

Abstract Remarkable progress in cancer immunology has revolutionized cancer therapy. The majority of patients, however, do not respond to immunotherapeutic options, warranting the ongoing search for better strategies. Leveraging the established role of protein tyrosine phosphatase non-receptor type 22 (PTPN22) in autoimmune diseases, we hypothesized that PTPN22 is a novel target for cancer immunotherapy. PTPN22 is a physiologic regulator of T cell receptor (TCR) signaling acting by dephosphorylating activating tyrosine residues in Lck and Zap70. We first confirmed the relevance of PTPN22 expression by exploring its expression in multiple human cancer types using The Cancer Genome Atlas (TCGA). PTPN22 expression positively correlated with T cell and M1 macrophage gene signatures and immune regulatory genes, especially inflamed tumor types. Next, we directly investigated the role of PTPN22 in antitumor immunity by comparing in vivo tumor characteristics in wild-type (WT) and PTPN22 knockout (KO) mice. Consistent with our hypothesis, PTPN22 KO mice resisted MC38 and EG7 tumors significantly compared with WT. Mass cytometry (CyTOF) profiling of the immune tumor microenvironment demonstrated that MC38 tumors in PTPN22 KO mice were infiltrated with greater numbers of T cells, particularly CD8+ T cells expressing granzyme B and PD1. To further delineate the effects of PTPN22 KO on TCR signaling, we established an optimized CyTOF panel of 9 phosphorylation sites involved in the TCR signaling pathway, including two enzymatic substrates of PTPN22 (Lck Y394 and Zap70 Y493) and 15 immune subtyping markers. CyTOF phospho-profiling of CD8 T cells from tumor-bearing mouse spleens and the peripheral blood of immunotherapy-naïve cancer patients showed that the phosphorylated state of Zap70 Y493 correlated strongly with granzyme B expression. Furthermore, phospho-profiling of tumor-infiltrating CD8+ T cells (a measure of T cell activation) revealed the highest TCR-pathway phosphorylation levels in memory CD8+ T cells that express PD1. The difference in phosphorylation levels between WT and PTPN22 KO was most pronounced for Lck Y394. Based on these findings, we then hypothesized that PD1 inhibition will further enhance the antitumor immune responses promoted by the lack of PTPN22. Indeed, PTPN22 KO mice bearing MC38 and EG7 tumors responded more significantly to anti-PD1 therapy when compared with tumor-bearing WT mice. Finally, we treated WT tumor bearing mice with two different small molecule inhibitors of PTPN22, one previously published compound, LTV1, and one novel compound, L1 (discovered through structure based synthesis). While both inhibitors phenocopied the PTPN22 KO mice in resisting MC38 tumor growth, L1 treatment gave an immune profile that resembled what was observed in tumor-bearing PTPN22 KO mice. Taken together, our results demonstrate that PTPN22 is a novel systemic target for augmenting antitumor immunity. Citation Format: Won Jin Ho, Jianping Lin, Ludmila Danilova, Zaw Phyo, Soren Charmsaz, Aditya Mohan, Todd Armstrong, Ben H. Park, Elana J. Fertig, Zhong-Yin Zhang, Elizabeth M. Jaffee. PTPN22 is a systemic target for augmenting antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3398.

In utero hypoxia altered Ang II-induced contraction via PKCβ in fetal cerebral arteries.

Cerebral circulation is important in fetal brain development, and angiotensin II (Ang II) plays vital roles in regulation of adult cerebral circulation. However, functions of Ang II in fetal cerebral vasculature and influences of in utero hypoxia on Ang II-mediated fetal cerebral vascular responses are largely unknown. This study investigated the effects and mechanisms of in utero hypoxia on fetal middle cerebral arteries (MCA) via Ang II. Near-term ovine fetuses were exposed to in utero hypoxia, and fetal MCA responses to Ang II were tested for vascular tension, calcium transient, and molecular analysis. Ang II caused significant dose-dependent contraction in control fetal MCA. Ang II-induced MCA constriction was decreased significantly in hypoxic fetuses. Neither losartan (AT1R antagonist, 10-5 mol/L) nor PD123,319 (AT2R antagonist, 10-5 mol/L) altered Ang II-mediated contraction in fetal MCA. Phenylephrine-mediated constriction was also significantly weaker in hypoxic fetuses. Bay K8644 caused similar contractions between the two groups. Protein expression of L-type voltage-dependent calcium channels was unchanged. There were no differences in caffeine-mediated vascular tension or calcium transients. Contraction induced by PDBu (PKC agonist) was obviously weaker in hypoxic MCA. Protein expression of PKCβ was reduced in the hypoxic compared with the control, along with no differences in phosphorylation levels. The results showed that fetal MCA was functionally responsive to Ang II near term. Intrauterine hypoxia reduced the vascular agonist-mediated contraction in fetal MCA, probably via decreasing PKCβ and its phosphorylation, which might play protective effects on fetal cerebral circulation against transient hypoxia.

The role of the TrkB-T1 receptor in the neurotrophin-4/5 antagonism of brain-derived neurotrophic factor on corticostriatal synaptic transmission.

This manuscript reviews the function and fundamental characteristics of the neurotrophins and their receptors to introduce the reader to the differential effects exhibited by the neurotrophins; brain-derived neurotrophic factor and neurotrophin 4/5 when acted together after sequential presentation. The neurotrophin 4/5 exhibits an inhibitory action on the modulatory effect of brain-derived neurotrophic factor in corticostriatal synapses when they are administered sequentially (brain-derived neurotrophic factor to neurotrophin 4/5). This inhibitory effect has not been previously documented and is relevant for these neurotrophins as both of them stimulate the TrkB receptor. The additive effect of these neurotrophins is also discussed and occurs when neurotrophin 4/5 exposure is followed by brain-derived neurotrophic factor in a mouse model of striatal degeneration. Occlusive and additive effects of both neurotrophins are accompanied by changes in the expression of the TrkB receptor isoforms, specifically TrkB-T1 and TrkB-FL, as well as differences in phosphorylation levels of the TrkB receptor. The results of the experiments described raise several questions to inquire about the role that TrkB-T1 receptor plays in striatal physiology, as well as the functional relevance of the interaction of brain-derived neurotrophic factor and neurotrophin 4/5 in the brain and more specifically at the striatal circuits in normal as well as pathological conditions.

Male-specific phosphorylated SR proteins in adult flies of the Mediterranean fruitfly Ceratitis capitata.

Alternative splicing is a widely used mechanism of gene regulation in sex determination pathways of Insects. In species from orders as distant as Diptera, Hymenoptera and Coleoptera, female differentiation relies on the activities of conserved splicing regulators, TRA and TRA-2, promoting female-specific expression of the global effector doublesex (dsx). Less understood is to what extent post-translational modifications of splicing regulators plays a role in this pathway. In Drosophila melanogaster phosphorylation of TRA, TRA-2 and the general RBP1 factor by the LAMMER kinase doa (darkener of apricot) is required for proper female sex determination. To explore whether this is a general feature of the pathway we examined sex-specific differences in phosphorylation levels of SR splicing factors in the dipteran species D. melanogaster, Ceratitis capitata (Medfly) and Musca domestica (Housefly). We found a distinct and reproducible pattern of male-specific phosphorylation on protein extracts enriched for SR proteins in C. capitata suggesting that differential phosphorylation may also contribute to the regulation of sex-specific splicing in the Medfly.

Elucidation of cellular signaling triggered by Francisella infection by comparative phosphoproteomic analysis (981.2)

Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune response that normally leads to protection against the infection. To elucidate the cellular signaling underlying this muted recognition of Francisella by the innate immune system, we performed a large‐scale comparative phosphoproteomic analysis of RAW 264.7 cells infected with either Francisella tularensis novicida or Salmonella Typhimurium, which is well recognized by the innate immune system. From our phosphoproteomics analysis >3000 phosphopeptides from >1200 phosphoproteins were identified, of which 368, 169, and 332 phosphopeptides were differentially abundant comparing Francisella‐ or Salmonella‐ infected and control cells at 0, 1, or 4 h post‐infection, respectively. Striking differences in phosphorylation levels on proteins from a diversity of cellular pathways were observed comparing Francisella and Salmonella infections, including key players of the innate immune response, such as AKT and MAP kinases and E3 ubiquitin ligases. In conclusion, our comparative phosphoproteomic analysis of cells infected with Francisella and Salmonella shows differences between the cellular signaling triggered by either pathogen, which may be reflective of specific mechanisms to evade the host immune response.Grant Funding Source: Supported by GM094623 and 8 P41 GM103493‐10

Analysis of the regulation of intracellular signaling pathways in peripheral blood cells in human studies

Introduction: Analysis of whole blood samples in human studies is a little-invasive method that provides direct information about the regulation of cell signaling avoiding the need for repeated tissue biopsies. However, the time interval and manipulations needed to eliminate erythrocytes from blood samples have the potential to introduce artifacts because phospho-epitopes respond to the environment and turn over rapidly. Our aim was to validate a procedure of quick fixation of whole blood samples suitable for subsequent analysis of phospho-epitopes of key signaling proteins. Methods: The first sample set included unfixed white blood cells treated with 1µM PMA for 10 min or sham-treated with medium. The second set included whole blood samples which were first PMA treated and then fixed and lysed by various protocols. We tested some available fix/lysis buffers or 4% formaldehyde solution combined with a range of red blood cell (RBC) lysis buffers (ice-cold water, ammonium chloride solution, Triton X-100 solution etc.). Finally, we compared protein concentrations in obtained samples and the phosphorylation of key signaling proteins using Western blotting, PathScan technology (Intracellular Signaling Array Kit, Cell Signaling) and Luminex technology (MILLIPLEX MAP Multi-pathway assays, Merck Millipore). Results: The final protocol employed the brief blood fixation with 4% formaldehyde, RBC lysis with 0.15% Triton X-100 and sample collection with protein lysis buffer containing 2% SDS. We found no significant differences in phosphorylation levels of key signaling proteins between samples unfixed or fixed by established method. In particular, an activation of MAPK, Akt, and mTOR signaling was successfully detected in PMA treated samples using PathScan and Luminex technology. Conclusion: We established a procedure of rapid fixation and isolation of peripheral blood cells for studying posttranslational modifications of intracellular signal proteins in human studies.

Potentiated B-Cell Antigen Receptor Signaling In Mantle Cell Lymphoma Is Associated With Overexpression Of Surface CD79B and IgM

BackgroundMantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma (NHL) and represents one of the most challenging lymphoma types to treat. The B-cell antigen Receptor (BCR) signaling pathway is increasingly implicated in the pathogenesis of some B-cell malignancies, and represents an attractive target for therapy. Recent clinical trials have shown that MCL is sensitive to small molecule inhibitors targeting BCR signaling, with response rates reported to be 68% for the BTK-inhibitor Ibrutinib, but only 40% for the PI3Kδ-inhibitor CAL-101. By contrast, chronic lymphocytic leukemia (CLL) has high response rates to both drugs. We hypothesized that basal- or BCR-induced phosphorylation of key molecules that transmit the BCR signal might differ across different NHL, and thereby help us understand therapy responses to small molecular inhibitors. MethodsSingle cell flow cytometry measurements of signaling were acquired for samples of MCL (n=34), all obtained prior to therapy. For comparison, samples from CLL (n=14), follicular lymphoma (FL, n=27), diffuse large B-cell lymphoma (DLBCL, n=12), as well as healthy donor peripheral blood (n=8) and tonsils (n=4) were included. BCR-induced signaling was obtained by stimulation with goat anti-human IgM F(ab')2 and IgG F(ab')2 fragments for 4 or 45 minutes. Phosphorylation of 14 signaling molecules was measured under basal (unstimulated) and activation-induced signaling, and CD20, CD3, CD5 and BCL2 were used for discrimination between malignant B cells and tumor infiltrating T cells. ResultsMeasurements of basal phosphorylation levels of signaling molecules displayed differences among the NHL types tested. MCL together with DLBCL had highest frequency of patients with tumor cells having elevated levels of p-p65 NF-kB. In contrast, CLL and FL did not have higher basal levels of p-p65 NF-kB. Elevated basal levels of p-Syk and p-AKT1 were most frequently found in CLL and DLBCL, and were less frequent in MCL and not found in FL. In addition, elevated basal level of p-SFK and p-STAT5 were found in a group of MCL patients. BCR-induced signaling in tumor cells showed striking differences across NHL types. For key BCR-signaling molecules including SFK, SYK and PLCg2, BCR-induced phosphorylation was significantly higher in MCL as compared to the other NHL, and was strikingly different from the low levels observed in CLL (Figure 1). Similar results were obtained for Src family kinases (SFK), ERK1/2, AKT1 and STAT5. Although BCR-induced signaling was highly potentiated in most MCL cases, there was a large variation within the MCL cohort. Hence, the fold change of BCR-induced p-PLCg2, relative to unstimulated cells, ranged from 0.28 to 3.01 in MCL tumors, as compared to 0.44 to 0.89 for healthy donor B cells. Furthermore, BCR-induced p-SYK in MCL correlated strongly with p-PLCg2 (r=0.96, p<0.0001), suggesting changes early in the signaling cascade. We therefore measured surface expression of the BCR subunits, IgM and CD79B, and found a strong association between BCR-induced p-SYK and IgM (Figure 1 r=0.65) and BCR-induced p-SYK and CD79B (r=0.64, p<0.0001). [Display omitted] ConclusionDifferences in basal phosphorylation levels of key signaling molecules were found across NHL. BCR-induced signaling was highly potentiated in MCL as compared to the other NHL, and was associated with increased expression of CD79B and IgM. Differences in phosphorylation levels of signaling molecules between different NHL, as well as heterogeneity within each entity might help us understand therapy responses to small molecular inhibitors, although this needs to be tested in follow-up studies. Disclosures:No relevant conflicts of interest to declare.

Nestin Protein Is Phosphorylated in Adult Neural Stem/Progenitor Cells and Not Endothelial Progenitor Cells

An intermediate filament protein, Nestin, is known as a neural stem/progenitor cell marker. It was shown to be required for the survival and self-renewal of neural stem cells according to the phenotypes of Nestin knockout mice. Nestin expression has also been reported in vascular endothelial cells, and we recently reported Nestin expression in proliferating endothelial progenitor cells, but not in mature endothelial cells. Using quantitative phosphoproteome analysis, we studied differences in phosphorylation levels between CNS Nestin in adult neural stem cells and vascular Nestin in adult bone-marrow-derived endothelial progenitor cells. We detected 495 phosphopeptides in the cell lysates of adult CNS stem/progenitor cells and identified 11 significant phosphorylated amino acid residues in the Nestin protein. In contrast, endothelial progenitor cells showed no significant phosphorylation of Nestin. We also measured neoplastic endothelial cells of the mouse brain and identified 13 phosphorylated amino acid residues in the Nestin protein. Among the 11 phosphorylated amino acids of adult CNS Nestin, five (S565, S570, S819, S883, and S886) were CNS Nestin-specific phosphorylation sites. Detection of the CNS-specific phosphorylation sites in Nestin, for example, by a phospho-specific Nestin antibody, may allow the expression of CNS Nestin to be distinguished from vascular Nestin.

Form of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A nonphosphorylated at tyrosine 145 and 147 is enriched in the nuclei of astroglial cells, adult hippocampal progenitors, and some cholinergic axon terminals

Compelling lines of evidence indicate that overexpression of dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) in subjects with trisomy 21 (Down syndrome[DS]) contributes to the abnormal structure and function of the DS brain. In the present study, we used a novel, phospho-dependent antibody recognizing DYRK1A only with nonphosphorylated tyrosine 145 and 147 (DYRK1A Tyr-145/147P(-)), to investigate the expression pattern of this DYRK1A species in trisomic and disomic human and mouse brains. Immunoblotting and dephosphorylation experiments demonstrated higher levels of DYRK1A Tyr-145/147P(-) in postnatal trisomic brains in comparison with controls (by ∼40%) than those of the DYRK1A visualized by three other N- and C-terminally directed antibodies to DYRK1A. By immunofluorescence, the immunoreactivity to DYRK1A Tyr-145/147P(-) was the strongest in the nuclei of astroglial cells, which contrasted with the predominantly neuronal localization of DYRK1A visualized by the three other antibodies to DYRK1A we used. In addition, DYRK1A Tyr-145/147P(-) was enriched in the nuclei of neuronal progenitors and newly born neurons in the adult hippocampal proliferative zone and also occurred in some cholinergic axonal terminals. Our data show a distinctive expression pattern of DYRK1A forms nonphosphorylated at Tyr-145 and Tyr-147 in the brain tissue and suggest that DS subjects may exhibit not only upregulation of total DYRK1A, but also more subtle differences in phosphorylation levels of this kinase in comparison with control individuals.

Hyperphosphorylation of Mouse Cardiac Titin Contributes to Transverse Aortic Constriction-Induced Diastolic Dysfunction

Mechanisms underlying diastolic dysfunction need to be better understood. To study the role of titin in diastolic dysfunction using a mouse model of experimental heart failure induced by transverse aortic constriction. Eight weeks after transverse aortic constriction surgery, mice were divided into heart failure (HF) and congestive heart failure (CHF) groups. Mechanical studies on skinned left ventricle myocardium measured total and titin-based and extracellular matrix-based passive stiffness. Total passive stiffness was increased in both HF and CHF mice, and this was attributable to increases in both extracellular matrix-based and titin-based passive stiffness, with titin being dominant. Protein expression and titin exon microarray analysis revealed increased expression of the more compliant N2BA isoform at the expense of the stiff N2B isoform in HF and CHF mice. These changes are predicted to lower titin-based stiffness. Because the stiffness of titin is also sensitive to titin phosphorylation by protein kinase A and protein kinase C, back phosphorylation and Western blot assays with novel phospho-specific antibodies were performed. HF and CHF mice showed hyperphosphorylation of protein kinase A sites and the proline glutamate valine lysine (PEVK) S26 protein kinase C sites, but hypophosphorylation of the PEVK S170 protein kinase C site. Protein phosphatase I abolished differences in phosphorylation levels and normalized titin-based passive stiffness levels between control and HF myocardium. Transverse aortic constriction-induced HF results in increased extracellular matrix-based and titin-based passive stiffness. Changes in titin splicing occur, which lower passive stiffness, but this effect is offset by hyperphosphorylation of residues in titin spring elements, particularly of PEVK S26. Thus, complex changes in titin occur that combined are a major factor in the increased passive myocardial stiffness in HF.

Preserving protein profiles in tissue samples: Differing outcomes with and without heat stabilization

Post translational modification (PTM) and proteolytic processing of proteins contributes to regulation of their stability, intracellular localization and interactions with other proteins, and to direct enhancement or repression of their activity. Both PTM and proteolysis are dynamic; levels and rates change in response to changes in the cellular environment. Tissue excision, post mortem interval and subsequent methods of tissue processing for protein analysis unavoidably alter the cellular environment and therefore features of protein profiles. To aid in understanding the time frame and protein specificity of these changes and the biological and technical contributions to them, we have compared features of protein profiles in mouse hippocampus and cortex following three methods of tissue handling: immediate lysate preparation, and rapid heating to 95 °C and standard snap freezing in liquid nitrogen, prior to lysate preparation. In spite of the very short time frames involved, we observe protein-specific differences in levels of phosphorylation, general differences in patterns of sumoylation, and specific differences in levels of proteolytic cleavage of calcineurin and the neurotrophin receptor, TRKA. These differences vary with brain region and with post excision time to processing, and highlight the challenges inherent in accurately profiling the in vivo proteome.

Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimen

Reversible phosphorylation of proteins is the most common PTM in cell-signaling pathways. Despite this, high-throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO2)-based 3-D LC (strong cationic exchange/TiO2/C18)-MS(3)-linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low-abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (H(2) (18)O for tissues and plasma). Two examples of potential HCC phospho-biomarkers including plectin-1(phopho-Ser-4253) and alpha-HS-glycoprotein (phospho-Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO2-based online-3-D LC-MS(3)-linear ion trap system with high-throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers.

Phosphorylation state of tau in the hippocampus of apolipoprotein E4 and E3 knock-in mice.

The apolipoprotein E (apoE) epsilon 4 allele is associated with an increased risk of sporadic as well as late-onset familial Alzheimer's disease (AD). To accurately determine the isoform-specific effects of human apoE on AD-like phosphorylation of tau, hippocampi from human apoE knock-in (KI) mice were studied by quantitative immunoblotting. There was no significant difference in phosphorylation levels of tau at nine of the 13 epitopes, for six of eight tau kinases, or in protein levels of three tau phosphatases, between apoE3-KI and apoE4-KI mouse hippocampi. However, in apoE4-KI mice, phosphorylation of tau at Ser235 was increased to approximately 150%, that at Ser413 to approximately 140%, while that at Ser202/Thr205 and Thr205 were decreased to approximately 70%, and the protein level of tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK3beta) was increased to approximately 120%, that of extracellular signal-regulated kinase 2 (ERK2) was increased to approximately 130%, compared with apoE3-KI mice.