Phospho-PAK2 (S141) Antibody
Description
Structure and Function of the Antibody
The Phospho-PAK2 (S141) Antibody is typically a rabbit polyclonal antibody (e.g., CSB-PA020222 from American Research Products) or monoclonal antibody (e.g., bsm-54514R from Bioss USA), raised against synthetic peptides corresponding to phosphorylated serine 141 of human PAK2. Its specificity ensures detection of PAK2 activation in various cell types, including human, mouse, and rat tissues . The antibody is often conjugated in a liquid format (e.g., 1 mg/ml in PBS with 50% glycerol) and requires storage at -20°C or -80°C to maintain stability .
Techniques
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Western Blot (WB): Detects a 65 kDa band corresponding to phosphorylated PAK2. Recommended dilutions: 1:300–1:5000 .
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Immunohistochemistry (IHC): Stains phosphorylated PAK2 in tissue sections (e.g., paraffin-embedded rat brain). Dilutions: 1:200–1:400 .
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Immunofluorescence (IF): Localizes activated PAK2 in cellular compartments .
Biological Contexts
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Apoptosis: PAK2 phosphorylation at S141 is linked to caspase-mediated cleavage, generating the pro-apoptotic PAK-2p34 fragment .
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Cell Proliferation: Required for EGF-induced cell growth via JUN phosphorylation .
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Cancer: Phosphorylated PAK2 correlates with tumor progression in breast and lung cancers .
PAK2 Activation Pathway
Phosphorylation at S141 occurs downstream of CDC42/RAC1 binding, inducing a conformational change that enables autophosphorylation and kinase activity . This activation regulates:
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Cytoskeleton Dynamics: PAK2 phosphorylates MAPKAPK5 to promote F-actin polymerization .
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Apoptosis: Caspase cleavage of PAK2 generates the PAK-2p34 fragment, which translocates to the nucleus to activate JNK signaling .
Disease Implications
Product Specs
Target Background
- Overexpression of PAK2 in oral squamous cell carcinomas is linked to advanced pathology grade. PMID: 29714078
- Human cytomegalovirusmiRUS45p promotes apoptosis in cells specifically through the inhibition of PAK2 expression. PMID: 28765936
- Leukemic cells require PAK2 for growth towards the extracellular matrix. PAK2-deficient cells fail to form colonies in methylcellulose and induce lymphomas in vivo. PAK2 might be the crucial isoform in leukemic cells by controlling tumor growth. PMID: 28707321
- PAK2 kinase exhibits an alternative anti-apoptotic role, phosphorylating caspase-7 and promoting unhindered cell growth and chemotherapeutic resistance. PMID: 27889207
- Overexpression of miR-137 inhibits melanoma cell proliferation, mimicking the effects of PAK2 knockdown using siRNAs. PMID: 26186482
- PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a potential target for rational drug therapy in TSC. PMID: 26412398
- Nef utilizes PAK2 in a stepwise mechanism where its kinase activity cooperates with an adaptor function for the exocyst complex to inhibit host cell actin dynamics. PMID: 26350970
- Cytoplasmic Pak2 may promote cell proliferation in normal endometrium during the menstrual cycle. PMID: 26218748
- HDAC6 may promote the growth of GBM cells by inhibiting SMAD2 phosphorylation to downregulate p21. PMID: 26150340
- Repression of microRNA miR-134 and subsequent up-regulation of p21-activated kinase 2 (Pak2) may contribute to paclitaxel resistance. PMID: 26363097
- Inhibition of PAK activation at late G2-phase centrosomes due to Rac1 inactivation coincides with impaired activation of Aurora A and the CyclinB/Cdk1 complex, delaying mitotic entry. PMID: 24840740
- Pak2 has been identified as a potential mediator of ovarian cancer cell migration on the extracellular matrix. PMID: 25050916
- PAK2 activation may be associated with advanced tumor progression and poor prognosis in gastric cancer. PMID: 24621074
- Prostasin represses cancer cells and contributes to chemoresistance by modulating the CASP/PAK2-p34/actin pathway. PMID: 24434518
- HIV-1 Nef expression mediates phosphorylation of Mek1 on serine298 and Pak2 on serine192/197 in T cell lines and primary human T cells. PMID: 23746211
- Thrombin induces monocyte/macrophage migration through PAR1-Galpha12-dependent Pyk2-mediated Gab1 and p115 RhoGEF interactions, leading to Rac1- and RhoA-targeted Pak2 activation. PMID: 24025335
- PAK2 negatively modulates TGF-beta signaling by attenuating the receptor-Smad interaction and subsequent Smad activation. PMID: 22393057
- Low-to-moderate penetrance protein coding mutations or non-coding mutations at DLG1 and/or PAK2, or a nearby gene, may reproduce the behavioral characteristics of the 3q29 microdeletion. PMID: 21850710
- The ability of Nef to associate with PAK2 correlates with its ability to enhance HIV-1 replication. PMID: 21819585
- Highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity. PMID: 21555521
- High PAK2 is associated with melanoma. PMID: 21177766
- Mechanistic studies of PAK2 autoactivation: a two-step model of cis initiation followed by trans amplification. PMID: 21098037
- Analysis of evolutionarily conserved residues crucial for the catalytic activity of PKA and Pak2. PMID: 20209159
- The association between the CD4 receptor and protein kinase pp58 and the protein-tyrosine kinase within the cell introduces a specific pathway for T lymphocyte activation. PMID: 20724730
- MYO18A is a novel binding partner of the PAK2/betaPIX/GIT1 complex, suggesting that MYO18A may play a significant role in regulating epithelial cell migration by affecting multiple cellular mechanisms. PMID: 19923322
- Knockdown of PAK2 enhances loss of cell-cell junctions and increases lamellipodium extension without affecting migration speed in Hepatocyte Growth Factor (HGF) stimulated DU145 prostate carcinoma cells. PMID: 19628037
- The enzymatic phosphorylation reaction of PAK2 can be best interpreted by a rapid-equilibrium random bi-bi reaction model; the catalysis reaction is partially limited by both the phosphoryl group transfer and the product release steps. PMID: 12549935
- The opposing effects of Core protein on the transcription of P21 might be significant in the progression of liver disease in HCV-positive patients. PMID: 12823590
- Caspase-activated PAK-2 is regulated by subcellular targeting and proteasomal degradation. PMID: 12853446
- Sites of PAK2 autophosphorylation in the regulatory and catalytic domains and their kinetic effect; multiple regions of PAK2 are involved in enzyme-substrate recognition. PMID: 12907671
- Pak2 phosphorylates Myc at three sites (T358, S373, and T400) and affects Myc functions both in vitro and in vivo. PMID: 14749374
- Nef induces signal transduction via the recruitment of a signaling machinery including Pak2 into lipid rafts, mimicking a physiological cellular mechanism to initiate the TCR cascade. PMID: 15047825
- PAK2 kinase activity is increased in response to TCR stimulation, suggesting a novel role for PAK2 as a positive regulator of T cell activation. PMID: 15187108
- PAK2 interacts with Nef proteins from SIV-infected chimpanzees. PMID: 15194762
- Pak2 plays a role in the down-regulation of translation initiation in apoptosis by phosphorylation of Mnk1. PMID: 15234964
- PS-GAP is a novel regulator of caspase-activated PAK-2. PMID: 15471851
- PAK-2 is activated in 1-LN prostate cancer cells by a proteinase inhibitor, alpha 2-macroglobulin. PMID: 15908432
- Binding of Cdc42 localizes Pak2 to the endoplasmic reticulum, where autophosphorylation alters the association of the two proteins. PMID: 16204230
- Pak2 binds to and phosphorylates initiation factor eIF4G, inhibiting the association of eIF4E with m7GTP, reducing translation initiation. PMID: 16281055
- Nef protein amino acids at positions 85, 89, 187, 188, and 191 (L, H, S, R, and F in the clade B consensus, respectively) are critical for Pak2 association and activation. PMID: 16501114
- Posttranslational myristoylation of PAK2 might be part of a unique series of mechanisms involved in the regulation of the later events of apoptosis. PMID: 16617111
- c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines. PMID: 16867995
- This study of tissue-derived HIV-1 Nefs demonstrates that CD4 and MHC-I downregulation are highly conserved Nef functions, while Pak2 association is variable in late-stage AIDS patients. PMID: 16979207
- Define a new class of PAK-interacting proteins, which play an important role in actin cytoskeletal reorganization. PMID: 17543336
- The interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis. PMID: 17881449
- Protein phosphatase 1alpha can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity. PMID: 18176785
- Data show RNAi-mediated or dominant-negative suppression of Pak2, major regulators of cytoskeletal signaling downstream of Cdc42 or Rac1, markedly inhibits EC lumen and tube formation. PMID: 18319301
- Huntingtin exerts anti-apoptotic effects by binding to Pak2, reducing the abilities of caspase-3 and caspase-8 to cleave Pak2 and convert it into a mediator of cell death. PMID: 19240112
- PAK-2 activity controls the apoptotic response by regulating levels of activated caspase 3 and thereby its own cleavage to the proapoptotic PAK-2p34 fragment. PMID: 19242610
Q&A
What validation strategies ensure specificity of Phospho-PAK2 (S141) antibodies in Western blotting?
Specificity validation requires a multi-pronged approach. First, phosphopeptide preabsorption is essential: incubating the antibody with the immunizing phosphopeptide should abolish signal, whereas dephosphorylated peptides should retain immunoreactivity . Second, enzymatic dephosphorylation of lysates via alkaline phosphatase treatment should eliminate the target band, as demonstrated in integrin signaling studies where PAK2 S141 phosphorylation was validated through phosphatase sensitivity . Third, genetic controls such as CRISPR-mediated PAK2 knockout or site-directed mutagenesis (e.g., S141A substitution) provide definitive evidence of antibody specificity, as shown in FLT3-dependent leukemia models where PAK2 phosphorylation was abolished by kinase inhibitors .
How does Phospho-PAK2 (S141) antibody performance vary across immunohistochemistry (IHC) and Western blotting (WB)?
Antigen retrieval and epitope stability are critical variables. For IHC, Tris-EDTA buffer (pH 9.0) optimizes phospho-epitope exposure in paraffin-embedded tissues, as validated in human tonsil sections . In contrast, WB requires boiling lysates in SDS-containing buffers to preserve labile phosphorylation states. Notably, PAK2 S141 phosphorylation exhibits tissue-specific stability; in lymphoid malignancies, fixation delays >24 hours significantly degrade signal . Parallel validation using siRNA-mediated PAK2 knockdown in target tissues (e.g., Jurkat T-cells) confirms antibody specificity across applications .
What controls are necessary when quantifying PAK2 activation dynamics?
Three-tiered controls are mandatory:
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Loading controls: Total PAK2 antibodies (e.g., ab76293) normalize for protein quantity .
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Pathway activity controls: Co-staining with phosphorylated substrates like MKNK1 or BAD validates functional kinase activity .
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Stimulation/Inhibition controls: Epidermal growth factor (EGF) stimulation (15 min) should increase S141 phosphorylation, while PAK inhibitors (e.g., FRAX486) reduce it .
How do conflicting reports on PAK2 S141 phosphorylation’s role in apoptosis resolution arise?
Discrepancies stem from isoform-specific compensation and cell type-dependent signaling. In FLT3-driven acute lymphoblastic leukemia (ALL), PAK2 S141 phosphorylation promotes survival via STAT5 activation, whereas caspase-cleaved PAK2 (PAK2p34) in epithelial cells induces apoptosis through JNK signaling . Experimental design must account for:
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Temporal resolution: Phospho-flow cytometry reveals S141 dynamics within 30–120 minutes post-integrin activation .
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Compensatory PAK1/PAK3 activity: Combined siRNA knockdown (PAK1 + PAK2) prevents false negatives, as shown in SEM leukemic cells .
Table 1: PAK2 S141 Functional Outcomes Across Models
What experimental frameworks resolve PAK2 S141 phosphorylation’s crosstalk with other kinases?
Phosphoproteomic integration with kinase inhibition profiling is optimal. In ALL models, TiO2-based phosphopeptide enrichment coupled with iTRAQ-MS identified PAK2 S141 as a hub for RTK/PAK crosstalk, with FLT3 and PDGFRB upstream regulation . To decouple direct vs. indirect effects:
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Kinase inhibitor panels: Midostaurin (FLT3 inhibitor) reduces S141 phosphorylation by 80% in XT-ALL22089 cells, whereas dasatinib (BCR-ABL inhibitor) has no effect .
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FRET-based biosensors quantify real-time PAK2 activation in live cells, circumventing fixation artifacts .
How do researchers address cross-reactivity with PAK1/PAK3 in multiplex assays?
Isoform-selective siRNA and knockout models are indispensable. In HEK293T cells, PAK1 silencing (siRNA) eliminates ab76293 cross-reactivity, confirming PAK2 specificity . For phospho-specific antibodies:
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Differential electrophoresis: PAK1 exhibits higher molecular weight (64–70 kDa) versus PAK2 (58–60 kDa) .
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Two-dimensional Phos-tag SDS-PAGE separates PAK2 S141 phosphorylation from PAK1 S144, resolving overlapping mobility shifts in glioblastoma models .
What protocols optimize Phospho-PAK2 (S141) detection in low-abundance samples?
Signal amplification via tyramide-based IHC or proximity ligation assays (PLA) enhances sensitivity. In T-LBL patient biopsies, PLA using Phospho-PAK2 (S141) and total PAK2 antibodies increased detection threshold 10-fold compared to conventional IHC . For WB:
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Immunoprecipitation: Anti-PAK2 magnetic beads (e.g., Dynabeads®) concentrate PAK2 from 2 mg lysate inputs .
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Extended exposure times: 30-minute exposures with chemiluminescent substrates (e.g., SuperSignal™) mitigate background in neuronal tissues .
How is Phospho-PAK2 (S141) data contextualized within broader signaling networks?
Pathway topology mapping using tools like MetaCore™ integrates PAK2 phosphorylation with interactome data. In FLT3-ITD ALL, PAK2 S141 phosphorylates ARHGEF6 and GIT1, linking it to cytoskeletal remodeling . Concurrently, Boolean network modeling predicts feedback loops where PAK2 inhibition upregulates PAK1 expression by 2.3-fold, necessitating dual targeting in therapeutic contexts .
