Phospho-AKT1 (T450) Recombinant Monoclonal Antibody
Description
Introduction to Phospho-AKT1 (T450) Recombinant Monoclonal Antibody
The Phospho-AKT1 (T450) Recombinant Monoclonal Antibody is a highly specific tool for detecting the phosphorylated form of AKT1 (Protein Kinase B alpha) at threonine 450 (T450). This antibody enables researchers to study the activation and regulation of AKT1, a serine/threonine kinase critical in signaling pathways governing cell survival, proliferation, and metabolism. AKT1 is activated through phosphorylation at T450 (and other residues like T308 and S473) downstream of PI3K signaling, which is frequently dysregulated in cancers, diabetes, and neurodegenerative diseases .
Role in Cell Cycle Regulation
Phosphorylation of AKT1 at T450 is cell-cycle-dependent, peaking during the G1/S phase. Studies demonstrate that cyclin A2/CDK2 complexes regulate AKT1 activation, which promotes tumor growth and survival . For example:
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Akt1-AA (T450/T308A mutant): Severely impaired phosphorylation and reduced tumor formation.
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Akt1-DE (T450/T308E mutant): Constitutively active, enhancing proliferation and tumorigenesis .
Pathological Implications
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Cancer: AKT1 hyperactivation via T450 phosphorylation drives apoptosis resistance and metastasis. Antibodies like ARC1524 are used to monitor AKT1 activation in breast and ovarian cancers .
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Metabolic Disorders: AKT1 regulates glucose uptake and glycogen synthesis. Dysregulation in T450 phosphorylation may contribute to insulin resistance .
Western Blotting
Antibodies such as EPR4157 (Abcam) and CABP0980 detect phosphorylated AKT1 in cell lysates (e.g., C6 glioma cells) . Key Protocol:
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Sample Preparation: Lyse cells, denature proteins, and separate via SDS-PAGE.
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Membrane Transfer: Transfer to PVDF/nitrocellulose.
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Blocking/Probing: Block with 5% milk/TBST; probe with primary antibody (1:500–1:2000) and HRP-conjugated secondary .
ELISA
EPR4157 shows high specificity in indirect ELISA, binding strongly to AKT1 (pT450) peptides but not non-phosphorylated or cross-reactive AKT2/3 isoforms .
Product Specs
The phospho-AKT1 (T450) recombinant monoclonal antibody is produced through a combination of protein technology and DNA recombinant techniques. Initially, an animal is immunized with a synthetic peptide derived from human phospho-AKT1 (T450), leading to the production of B cells. Phospho-AKT1 (T450) antibody-producing B cells are then selected and subjected to single clone identification. The genes encoding the phospho-AKT1 (T450) antibody are amplified using PCR and inserted into a plasmid vector to create a recombinant vector. This vector is introduced into host cells for antibody expression. The phospho-AKT1 (T450) recombinant monoclonal antibody is purified from the cell culture supernatant using affinity chromatography. It exhibits specificity for human AKT1 phosphorylated at T450 residue. Rigorous validation procedures ensure its accuracy and suitability for ELISA and WB applications.
Target Background
AKT1 is one of three closely related serine/threonine-protein kinases (AKT1, AKT2, and AKT3) collectively known as the AKT kinase. AKT kinases play a crucial role in regulating various cellular processes, including metabolism, proliferation, cell survival, growth, and angiogenesis. This regulation is achieved through serine and/or threonine phosphorylation of a diverse range of downstream substrates. Over 100 substrate candidates have been reported to date, although isoform specificity remains undefined for most of them.
AKT1 is responsible for regulating glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface. Phosphorylation of PTPN1 at 'Ser-50' negatively modulates its phosphatase activity, preventing dephosphorylation of the insulin receptor and attenuating insulin signaling. Phosphorylation of TBC1D4 triggers the binding of this effector to inhibitory 14-3-3 proteins, a necessary step for insulin-stimulated glucose transport. AKT1 also regulates the storage of glucose in the form of glycogen by phosphorylating GSK3A at 'Ser-21' and GSK3B at 'Ser-9', leading to inhibition of its kinase activity.
Phosphorylation of GSK3 isoforms by AKT1 is also believed to contribute to cell proliferation. AKT1 further regulates cell survival through phosphorylation of MAP3K5 (apoptosis signal-related kinase). Phosphorylation of 'Ser-83' reduces MAP3K5 kinase activity stimulated by oxidative stress, thereby preventing apoptosis. AKT1 mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', activating mTORC1 signaling and leading to both phosphorylation of 4E-BP1 and activation of RPS6KB1.
AKT1 is involved in the phosphorylation of members of the FOXO factors (Forkhead family of transcription factors), resulting in binding of 14-3-3 proteins and cytoplasmic localization. Notably, FOXO1 is phosphorylated at 'Thr-24', 'Ser-256', and 'Ser-319'. Equivalent sites on FOXO3 and FOXO4 are also phosphorylated. AKT1 plays a significant role in regulating NF-kappa-B-dependent gene transcription and positively regulates the activity of CREB1 (cyclic AMP (cAMP)-response element binding protein). Phosphorylation of CREB1 induces the binding of accessory proteins essential for the transcription of pro-survival genes such as BCL2 and MCL1.
AKT1 phosphorylates 'Ser-454' on ATP citrate lyase (ACLY), potentially regulating ACLY activity and fatty acid synthesis. It activates the 3B isoform of cyclic nucleotide phosphodiesterase (PDE3B) via phosphorylation of 'Ser-273', leading to reduced cyclic AMP levels and inhibition of lipolysis. AKT1 phosphorylates PIKFYVE on 'Ser-318', resulting in increased PI(3)P-5 activity. The Rho GTPase-activating protein DLC1 is another substrate, and its phosphorylation is implicated in the regulation of cell proliferation and cell growth.
AKT1 serves as a key modulator of the AKT-mTOR signaling pathway, controlling the tempo of newborn neuron integration during adult neurogenesis. This includes proper neuron positioning, dendritic development, and synapse formation. AKT1 signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, and insulin-like growth factor I (IGF-I). AKT1 mediates the antiapoptotic effects of IGF-I. It is essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
AKT1 may be involved in regulating placental development. It phosphorylates STK4/MST1 at 'Thr-120' and 'Thr-387', leading to inhibition of its kinase activity, nuclear translocation, autophosphorylation, and ability to phosphorylate FOXO3. AKT1 also phosphorylates STK3/MST2 at 'Thr-117' and 'Thr-384', leading to inhibition of its cleavage, kinase activity, autophosphorylation at Thr-180, binding to RASSF1, and nuclear translocation. It further phosphorylates SRPK2, enhancing its kinase activity towards SRSF2 and ACIN1 and promoting its nuclear translocation.
AKT1 phosphorylates RAF1 at 'Ser-259' and negatively regulates its activity. Phosphorylation of BAD stimulates its pro-apoptotic activity. AKT1 phosphorylates KAT6A at 'Thr-369', inhibiting the interaction of KAT6A with PML and negatively regulating its acetylation activity towards p53/TP53. It phosphorylates palladin (PALLD), modulating cytoskeletal organization and cell motility. AKT1 phosphorylates prohibitin (PHB), playing an important role in cell metabolism and proliferation. It phosphorylates CDKN1A, where phosphorylation at 'Thr-145' induces its release from CDK2 and cytoplasmic relocalization. These recent findings suggest that the AKT1 isoform plays a more specific role in cell motility and proliferation.
AKT1 phosphorylates CLK2, thereby controlling cell survival in response to ionizing radiation. It phosphorylates PCK1 at 'Ser-90', reducing the binding affinity of PCK1 to oxaloacetate and transforming PCK1 into an atypical protein kinase activity using GTP as a donor. AKT1 also acts as an activator of TMEM175 potassium channel activity in response to growth factors. It forms the lysoK(GF) complex together with TMEM175 and promotes TMEM175 channel activation, independently of its protein kinase activity.
- The optimal melatonin concentration (3 mM) significantly decreased the intracellular reactive oxygen species levels, caspase-3 activity, and the percentage of both dead and apoptotic-like sperm cells. Additionally, it increased vitality, progressive motility, total motility, and AKT phosphorylation compared with the control group. PMID: 29196809
- The findings indicate that SPRY4 and SPRY4-IT1 may act as oncogenes in testicular germ cell tumors via activation of the PI3K/Akt signaling pathway. PMID: 29410498
- Results suggest that transient receptor potential vanilloid 4 (TRPV4) accelerates glioma migration and invasion through the AKT/Rac1 signaling, and TRPV4 might be considered as a potential target for glioma therapy. PMID: 29928875
- Data indicate a regulatory mechanism underlying drug resistance and suggest that tribbles homologue 2 (TRIB2) functions as a regulatory component of the PI3K network, activating AKT in cancer cells. PMID: 28276427
- Findings indicated that shikonin inhibits proliferation and promotes apoptosis in human endometrioid endometrial cancer (EEC) cells by modulating the miR-106b/PTEN/AKT/mTOR signaling pathway, suggesting shikonin could act as a potential therapeutic agent in the EEC treatment. PMID: 29449346
- SIRT6 inhibited proliferation, migration, and invasion of colon cancer cells by up-regulating PTEN expression and down-regulating AKT1 expression. PMID: 29957460
- LHPP suppresses cell proliferation and metastasis in cervical cancer, and promotes apoptosis by suppressing AKT activation. PMID: 29944886
- Data show that activated proto-oncogene protein Akt (AKT) directly phosphorylates Fas associated factor 1 (FAF1), reduces FAF1 at the plasma membrane, and results in an increase in TGF-beta type II receptor (TbetaRII) at the cell surface. PMID: 28443643
- Data show that while overexpression of AKT serine/threonine kinase 1 (AKT1) promoted local tumor growth, downregulation of AKT1 or overexpression of AKT serine/threonine kinase 2 (AKT2) promoted peritumoral invasion and lung metastasis. PMID: 28287129
- High AKT1 expression is associated with metastasis in ovarian cancer. PMID: 29739299
- Circ-CFH promotes glioma progression by sponging miR-149 and regulating the AKT1 signaling pathway. PMID: 30111766
- High AKT1 expression is associated with metastasis via epithelial-mesenchymal transition carcinoma in colorectal cancer. PMID: 30066935
- High AKT1 expression is associated with tumor-node-metastasis in non-small cell lung cancer. PMID: 30106450
- High expression of AKT1 is associated with drug resistance and proliferation of breast cancer. PMID: 28165066
- Germline variants in the AKT1 gene are associated with prostate cancer. PMID: 29298992
- High AKT1 expression is associated with cisplatin-resistant oral cancer. PMID: 29956797
- Akt1 was identified as a novel target for miR-637, and its knockdown also induced cell growth inhibition and apoptosis in pancreatic ductal adenocarcinoma cells. PMID: 29366808
- High AKT1 expression is associated with periodontitis. PMID: 30218719
- High AKT1 expression is associated with angiogenesis of esophageal squamous cell carcinoma. PMID: 30015941
- High AKT1 expression is associated with Pancreatic Ductal Adenocarcinoma Metastasis. PMID: 29386088
- In MCF-7 cells, AIB1 overexpression increases p-AKT (Ser 473) activity. In both T47D and MCF-7 cells overexpressing A1B1, p-AKT (Ser 473) expression was significantly increased in the presence or absence of IGF-1, but increased more in the presence of IGF-1. PMID: 29808803
- In this study, the Ion Personal Genome Machine (PGM) and Ion Torrent Ampliseq Cancer panel were used to sequence hotspot regions from PIK3CA, AKT, and PTEN genes to identify genetic mutations in 39 samples of TNBC subtype from Moroccan patients and to correlate the results with clinical-pathologic data. PMID: 30227836
- The AKT pathway is activated by CBX8 in hepatocellular carcinoma. PMID: 29066512
- A direct interaction of both MEK1 and MEK2 with AKT was identified. The interaction between MEK and AKT affects cell migration and adhesion, but not proliferation. The specific mechanism of action of the MEK-AKT complex involves phosphorylation of the migration-related transcription factor FoxO1. PMID: 28225038
- miR-195 suppresses cell proliferation of ovarian cancer cells through regulation of VEGFR2 and AKT signaling pathways. PMID: 29845300
- High AKT1 expression is associated with cell growth, aggressiveness, and metastasis in gastric cancer. PMID: 30015981
- This is the first report showing that long-duration exposure to nicotine causes increased proliferation of human kidney epithelial cells through activation of the AKT pathway. PMID: 29396723
- RBAP48 overexpression contributes to the radiosensitivity of AGS gastric cancer cells via phosphoinositide 3-kinase/protein kinase B pathway suppression. PMID: 29901205
- Activating Akt1 mutations alter DNA double-strand break repair and radiosensitivity. PMID: 28209968
- PI3K-Akt pathway inhibitors, Akti-1/2 and LY294002, reduced PFKFB3 gene induction by PHA, as well as Fru-2,6-P2 and lactate production. Moreover, both inhibitors blocked activation and proliferation in response to PHA, demonstrating the importance of the PI3K/Akt signaling pathway in the antigen response of T-lymphocytes. PMID: 29435871
- RIO kinase 3 (RIOK3) positively regulates the activity of the AKT/mTOR pathway in glioma cells. PMID: 29233656
- High AKT1 phosphorylation is associated with colorectal carcinoma. PMID: 29970694
- Results show that AKT1 was associated with hypertension in Mexican Mestizos but not Mexican Amerindians. PMID: 30176313
- TERT could induce thyroid carcinoma cell proliferation mainly through the PTEN/AKT signaling pathway. PMID: 29901196
- Findings uncover a new function of p53 in the regulation of Akt signaling and reveal how p53, ASS1, and Akt are interrelated. PMID: 28560349
- Quantitative mass spectrometry of IAV1918-infected cells was performed to measure host protein dysregulation. Selected proteins were validated by immunoblotting, and phosphorylation levels of members of the PI3K/AKT/mTOR pathway were assessed. PMID: 29866590
- Radiation resistance tumors have upregulated Onzin and POU5F1 expression. PMID: 29596836
- The essential role of AKT in endocrine therapy resistance in estrogen receptor-positive, HER2-negative breast cancer is reviewed. PMID: 29086897
- FAL1 may work as a ceRNA to modulate AKT1 expression via competitively binding to miR-637 in HSCR. PMID: 30062828
- The overexpression of CHIP significantly increased the migration and invasion of DU145 cells, possibly due to activation of the AKT signaling pathway and upregulation of vimentin. The expression level of CHIP was observed to be increased in human prostate cancer tissues compared with the adjacent normal tissue. PMID: 29693147
- Genistein (GE) inhibited the growth of human Cholangiocarcinoma (CCA) cell lines by reducing the activation of EGFR and AKT, and by attenuating the production of IL6. E2 and ER were also involved in the growth-inhibitory effect of GE in CCA cells. PMID: 29693152
- This study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation. PMID: 29947926
- The role of USP18 in breast cancer provides a novel insight into the clinical application of the USP18/AKT/Skp2 pathway. PMID: 29749454
- Collectively, these results indicate that COX-1/PGE2/EP4 upregulates the beta-arr1 mediated Akt signaling pathway to provide mucosal protection in colitis. PMID: 28432343
- The AKT kinase pathway is regulated by SPC24 in breast cancer. PMID: 30180968
- CREBRF promotes the proliferation of human gastric cancer cells via the AKT signaling pathway. PMID: 29729692
- These results indicate that miR124 transection inhibits the growth and aggressiveness of osteosarcoma, potentially via suppression of TGFbeta-mediated AKT/GSK3beta/snail family transcriptional repressor 1 (SNAIL1) signaling, suggesting miR124 may be a potential anticancer agent/target for osteosarcoma therapy. PMID: 29488603
- Piperine reduced the expression of pAkt, MMP9, and pmTOR. Together, these data indicated that piperine may serve as a promising novel therapeutic agent to better overcome prostate cancer metastasis. PMID: 29488612
- S100A8 gene knockdown reduced cell proliferation in HEC-1A cells compared with control cells, induced cell apoptosis, inhibited the phosphorylation of protein kinase B (Akt), and induced the expression of pro-apoptotic genes. PMID: 29595187
- Intact keratin filaments are regulators for PKB/Akt and p44/42 activity, both basal and in response to stretch. PMID: 29198699
Q&A
What is the significance of T450 phosphorylation in AKT1 function?
T450 phosphorylation serves as a priming step in the multi-stage activation process of AKT1. Specifically, AKT1 is initially phosphorylated at Thr-450 by JNK kinases, which prepares the protein for subsequent phosphorylation events . This initial phosphorylation is crucial for protein stability, as evidenced by studies showing that absence of turn motif phosphorylation leads to significantly reduced protein stability and decreased kinase activity .
When properly phosphorylated at T450, AKT1 maintains its structural integrity and is positioned for further activation through phosphorylation at T308 (by PDK1) and S473 (by various kinases including PKD2) . The importance of T450 phosphorylation in regulating the stability of AKT1 and other AGC kinases is well-established in the scientific literature .
How does T450 phosphorylation compare to other phosphorylation sites on AKT1?
AKT1 activation involves a precisely orchestrated series of phosphorylation events at different sites, each serving distinct regulatory functions:
| Phosphorylation Site | Kinase Responsible | Primary Function | Position in Activation Sequence |
|---|---|---|---|
| T450 | JNK kinases | Priming and protein stability | First step |
| T308 | PDK1 | Necessary for maximal signaling | Second step |
| S473 | Various (including PKD2) | Increased catalytic activity with certain substrates | Final step |
What are the standard protocols for detecting Phospho-AKT1 (T450)?
Detection of phospho-AKT1 (T450) typically relies on antibody-based methods. When selecting experimental approaches, researchers should consider:
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Western Blotting: The most common method for detecting phospho-specific forms of AKT1. Protocols typically involve:
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Cell lysis under phosphatase inhibition conditions
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Protein separation by SDS-PAGE
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Transfer to membranes
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Blocking and probing with phospho-specific T450 antibodies
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Detection via chemiluminescence or fluorescence systems
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Dot Blotting: A validated application for phospho-AKT1 (T450) antibodies where 50ng of phospho-peptide or non-phospho-peptide are adsorbed to nitrocellulose membrane, and antibodies are applied at working concentrations of approximately 0.6μg per ml .
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Immunohistochemistry/Immunofluorescence: Can be used for tissue or cellular localization studies of phosphorylated AKT1.
Careful sample preparation is essential, particularly the inclusion of phosphatase inhibitors to prevent dephosphorylation during extraction procedures.
How does phospho-form specific substrate selection by AKT1 influence experimental design?
The phosphorylation status of AKT1 globally regulates its substrate specificity, creating significant implications for experimental design . Research has demonstrated that different phospho-forms of AKT1 display distinct preferences for substrates, with S473 phosphorylation particularly influencing substrate selectivity.
When designing experiments to study AKT1 function, researchers should consider:
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Phospho-form Isolation: Methods to produce consistent preparations of individual phospho-forms have been developed, allowing more precise investigation of substrate preferences than commercial preparations from Sf9 insect cells that contain variable mixtures of active AKT1 phospho-forms .
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Substrate Selection: AKT1 phosphorylated at S473 displays selectivity for particular substrates that is distinct from the doubly phosphorylated (T308 and S473) enzyme . Therefore, when studying potential new AKT1 substrates, researchers should evaluate activity with each phospho-form separately.
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Kinetic Analysis: Kinase activity assays with potential substrate peptides should be benchmarked against known substrates (e.g., GSK-3β) to accurately assess relative phosphorylation efficiency .
What are the technical challenges in generating and validating phospho-specific AKT1 antibodies?
Developing highly specific phospho-AKT1 (T450) antibodies presents several technical challenges:
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Peptide Design: The antibody production typically begins with a KLH conjugated synthetic phosphopeptide corresponding to amino acid residues surrounding T450 of human AKT1 . The design must ensure the phosphopeptide captures the appropriate structural context while maintaining specificity.
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Cross-reactivity Assessment: Validation must include rigorous testing against non-phosphorylated forms and closely related phospho-sites (particularly T308 and S473) to ensure specificity.
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Validation Methodology: Comprehensive validation should include:
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Dot blot analysis comparing phospho-peptide vs. non-phospho-peptide reactivity
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Western blot validation using samples with known phosphorylation states
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Phosphatase treatment controls to confirm phospho-specificity
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Testing across multiple cell types and conditions
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Reproducibility Challenges: Batch-to-batch variation must be carefully controlled, particularly for polyclonal antibodies where variability can significantly impact experimental outcomes.
How do PP-1 mediated dephosphorylation mechanisms impact AKT1 (T450) experimental interpretation?
Protein serine/threonine phosphatase-1 (PP-1) has been identified as a major phosphatase that directly dephosphorylates AKT at Thr-450, adding complexity to experimental design and interpretation . When studying AKT1 phosphorylation dynamics, researchers must consider:
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Phosphatase Inhibition Strategy: During protein extraction, appropriate phosphatase inhibitors must be selected that effectively inhibit PP-1 activity to preserve the native phosphorylation state of T450.
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PP-1 Isoform Specificity: Research has demonstrated that stable knockdown of PP-1α or PP-1β, but not PP-1γ, leads to enhanced phosphorylation of AKT at Thr-450 . This isoform specificity should inform experimental design and interpretation.
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Functional Consequences: Dephosphorylation of AKT by PP-1 significantly modulates its functions in:
These functional impacts must be considered when interpreting phenotypic results in AKT1 signaling studies.
What are the optimal expression systems for producing phospho-AKT1 (T450) for structural and functional studies?
Selection of an appropriate expression system is critical for studies requiring well-defined phosphorylated AKT1:
| Expression System | Phosphorylation Profile | Advantages | Limitations |
|---|---|---|---|
| Baculovirus-infected insect cells | Stoichiometric T450, substoichiometric T308 (<5%) and S473 (<0.5%) | Near-complete T450 phosphorylation | Mixed phosphorylation at other sites |
| Expressed protein ligation | Site-specific phosphorylation possible | Control over phosphorylation status | Complex methodology, potential for incomplete T450 phosphorylation |
| E. coli co-expression with kinases | Variable | Scalable production | Inconsistent phosphorylation |
For studies requiring precise control over multiple phosphorylation sites, expressed protein ligation methods have been developed, though careful monitoring of phosphorylation status is essential as incomplete T450 phosphorylation has been observed in some preparations .
How should researchers approach contradictory findings regarding phospho-AKT1 (T450) functions in different contexts?
Contradictory findings regarding phospho-AKT1 (T450) functions are not uncommon, particularly across different tissue types or disease contexts. A systematic approach to resolving such contradictions includes:
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Cell/Tissue Type Considerations: AKT1 function can vary significantly between tissue types. For example, in pancreatic cancer, higher expression of p-Akt1 has been correlated with favorable prognosis (23.0 vs. 9.3 months survival) , which contrasts with findings in some other cancer types.
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Methodological Standardization:
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Antibody validation: Ensure antibodies are detecting the same epitope and phosphorylation state
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Sample preparation: Standardize lysis conditions and phosphatase inhibition
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Quantification methods: Use consistent approaches for normalization and quantification
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Integration of Multiple Techniques:
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Biochemical assays to measure kinase activity
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Genetic approaches (knock-in mutations of T450 to non-phosphorylatable residues)
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Structural studies to understand conformational changes
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Systems biology approaches to map network effects
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Context-Specific Analysis: Carefully document experimental conditions, cell cycle status, and upstream activators/inhibitors that may explain divergent findings.
How can phospho-AKT1 (T450) status be leveraged for cancer research applications?
The phosphorylation status of AKT1 at T450 has significant implications for cancer research:
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Prognostic Biomarker Development: High p-Akt1 expression has been associated with favorable prognosis in certain cancers, such as pancreatic cancer, where it correlates with lower T stage . Researchers can investigate T450 phosphorylation as part of a phosphorylation signature that might predict treatment response or patient outcomes.
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Therapeutic Resistance Mechanisms: AKT activation has been associated with chemotherapy and radiotherapy resistance in several human cancers . Understanding the specific contribution of T450 phosphorylation to this resistance could inform combination therapy approaches.
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Drug Development Strategies:
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Targeting upstream kinases (JNK) that phosphorylate T450
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Developing agents that modulate PP-1 activity to alter T450 phosphorylation
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Creating conformation-specific inhibitors that recognize different phospho-states
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Experimental Models: Developing knock-in models with phosphomimetic or phospho-deficient mutations at T450 can help dissect the specific contributions of this phosphorylation site to cancer progression.
What experimental controls are essential when studying the relationship between AKT1 T450 phosphorylation and substrate selection?
Robust experimental design for studying phospho-T450 AKT1 substrate selection requires multiple controls:
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Phosphorylation-State Controls:
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Non-phosphorylated AKT1
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Single-site phosphorylated forms (pT450, pT308, pS473)
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Multi-site phosphorylated forms (pT450/pT308, pT450/pS473, pT308/pS473, pT450/pT308/pS473)
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Kinase Activity Validation:
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ATP binding affinity measurements (KM determinations)
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Positive control substrate (GSK-3β peptide) to normalize activity across preparations
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Enzyme concentration standardization based on activity rather than protein quantity
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Substrate Specificity Controls:
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Validation with peptides representing predicted AKT1 substrates based on OPAL data
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Mutated substrate sequences (alanine scanning) to confirm specificity determinants
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Competition assays with known substrates
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Biological Validation:
