Phospho-PAK2 (Ser141) Antibody

What is PAK2 and what is the significance of its phosphorylation at Ser141?

PAK2 (p21-activated kinase 2) is a serine/threonine protein kinase that functions as a downstream effector of the small GTPases CDC42 and RAC1. The protein has a calculated molecular weight of approximately 60 kDa and is encoded by the gene with NCBI ID 5062 . PAK2 plays critical roles in numerous cellular processes including cytoskeleton regulation, cell motility, cell cycle progression, and the balance between apoptosis and proliferation .

Phosphorylation at Ser141 is one of several key phosphorylation events in PAK2 activation. This site is part of the regulatory mechanism that controls PAK2 kinase activity. When small GTPases like CDC42 and RAC1 bind to PAK2, they induce a conformational change that leads to autophosphorylation at multiple sites, including Ser141 . This phosphorylation event contributes to the stability of the active conformation of PAK2, allowing it to phosphorylate downstream targets including MAPK4, MAPK6, JUN, histone H4, and various other substrates involved in cellular signaling networks .

How do Phospho-PAK2 (Ser141) antibodies work in experimental systems?

Phospho-PAK2 (Ser141) antibodies are specifically designed to recognize and bind to PAK2 only when it is phosphorylated at the serine 141 residue. These antibodies are typically generated by immunizing host animals (commonly rabbits) with a synthetic phosphopeptide corresponding to the sequence surrounding the Ser141 phosphorylation site of human PAK2 . The specific immunogen used is often a peptide with the sequence Y-L-S(p)-F-T, where S(p) represents the phosphorylated serine residue .

The highly specific nature of these antibodies allows researchers to distinguish between the phosphorylated (active) and non-phosphorylated (inactive) forms of PAK2. This specificity is achieved through rigorous purification processes and validation to ensure minimal cross-reactivity with non-phosphorylated PAK2 or other phosphorylated proteins . In experimental applications, these antibodies bind to their target epitope with high affinity, enabling detection through various secondary detection methods, including fluorophore-conjugated or enzyme-linked secondary antibodies depending on the experimental readout required .

What are the recommended experimental applications for Phospho-PAK2 (Ser141) antibodies?

Phospho-PAK2 (Ser141) antibodies are versatile tools that can be employed in multiple experimental contexts. The primary recommended application is Western blotting, typically at dilutions ranging from 1:500 to 1:3000, depending on the specific antibody formulation and experimental conditions . Western blotting allows researchers to assess changes in PAK2 phosphorylation status in response to various stimuli, inhibitors, or genetic manipulations.

These antibodies can also be utilized in cell-based ELISA assays, which provide a platform for detecting and quantifying Phospho-PAK2 (Ser141) in cultured cells . In these assays, the phosphorylated protein is captured by the Phospho-PAK2 (Ser141) antibody and then detected using HRP-conjugated secondary antibodies, allowing for colorimetric readout . This approach enables researchers to assess the effects of different stimulation conditions on PAK2 phosphorylation across various cell lines.

Multiple normalization methods can be employed when using these antibodies in quantitative applications:

• Using GAPDH antibodies as internal positive controls

• Crystal Violet whole-cell staining to normalize for cell density

• Using non-phospho-specific PAK2 antibodies to normalize for total PAK2 expression levels

What is the difference between phosphorylated and non-phosphorylated PAK2 in terms of function?

The phosphorylation status of PAK2 dramatically alters its functional capabilities within cellular signaling networks. Non-phosphorylated PAK2 exists primarily in an inactive conformation, wherein an autoinhibitory domain blocks the kinase domain, preventing catalytic activity . In this state, PAK2 is unable to phosphorylate downstream substrates.

Upon activation by GTPases like CDC42 and RAC1, PAK2 undergoes a conformational change that initiates autophosphorylation at multiple sites, including Ser141 . This phosphorylation stabilizes the protein in its active conformation, unleashing its kinase activity. Fully activated PAK2 can then phosphorylate numerous downstream targets involved in:

• Cytoskeletal reorganization and cell motility through MAPK4, MAPK6, and the downstream target MAPKAPK5, which regulates F-actin polymerization

• Cell proliferation via phosphorylation of JUN, particularly in EGF-induced proliferation pathways

• Regulation of chromatin structure through histone H4 phosphorylation, promoting assembly of H3.3 and H4 into nucleosomes

• Inhibition of apoptosis by phosphorylating CASP7, thereby preventing its activity

Full-length phosphorylated PAK2 generally promotes cell survival and growth, contrasting with the pro-apoptotic function of its caspase-cleaved fragment, PAK-2p34 .

How specific are Phospho-PAK2 (Ser141) antibodies compared to other PAK family antibodies?

The PAK family consists of six members divided into two groups. PAK1, PAK2, and PAK3 comprise Group I, and these proteins share significant sequence homology, particularly around key phosphorylation sites . For instance, Ser141 in PAK2 corresponds to Ser144 in PAK1, and these sites are surrounded by similar amino acid sequences . Consequently, some antibodies may detect both Phospho-PAK1 (Ser144) and Phospho-PAK2 (Ser141), as seen with the antibody described in search result , which recognizes PAK1, PAK2, and PAK3 when phosphorylated at their respective serine residues.

For research requiring absolute specificity for Phospho-PAK2 (Ser141), careful antibody selection and validation are essential. Researchers should review the epitope sequence used for immunization and conduct appropriate control experiments to confirm specificity for their particular experimental system .

What are recommended storage conditions and stability considerations for Phospho-PAK2 (Ser141) antibodies?

Proper storage and handling of Phospho-PAK2 (Ser141) antibodies are crucial for maintaining their specificity and activity over time. The typical recommended storage condition for these antibodies is 4°C in the dark for up to 6 months . For conjugated antibodies (e.g., biotin or fluorophore-labeled), protection from light is particularly important to prevent photobleaching of the conjugated molecules.

Most Phospho-PAK2 (Ser141) antibodies are formulated in buffer solutions containing stabilizers and preservatives. A common formulation includes:

• 0.01M Sodium Phosphate

• 0.25M NaCl

• pH 7.6

• 5mg/ml Bovine Serum Albumin

• 0.02% Sodium Azide

The inclusion of BSA helps stabilize the antibody, while sodium azide prevents microbial contamination. When working with these antibodies, researchers should avoid repeated freeze-thaw cycles, which can lead to protein denaturation and loss of antibody activity. If longer storage is required, aliquoting the antibody and storing at -20°C or -80°C may be considered, though specific recommendations may vary between manufacturers.

How does the autophosphorylation mechanism of PAK2 at Ser141 relate to phosphorylation at other sites like Thr402?

PAK2 activation involves a complex, multi-step phosphorylation cascade with distinct mechanisms governing different phosphorylation sites. Research indicates that while both Ser141 and Thr402 are critical phosphorylation sites in PAK2, they serve different roles in the activation process and are regulated through different mechanisms .

Autophosphorylation of Thr402, located in the activation loop of PAK2's catalytic domain, follows a two-step mechanism: cis initiation followed by trans amplification . Initially, unphosphorylated PAK2 undergoes an intramolecular (cis) autophosphorylation on Thr402 to produce minimally active phosphorylated PAK2. This newly formed active PAK2 then phosphorylates other PAK2 molecules at Thr402 in an intermolecular (trans) manner, amplifying the activation signal . This mechanism has been quantitatively characterized using kinetic approaches that can distinguish between cis- and trans-pathways in autocatalytic reactions.

In contrast, phosphorylation at Ser141 appears to be regulated differently and may precede or follow Thr402 phosphorylation depending on the activation context. While Thr402 phosphorylation is essential for catalytic activity, Ser141 phosphorylation may play more of a role in stabilizing the active conformation or regulating interactions with specific binding partners . The interplay between these phosphorylation events creates a sophisticated regulatory network that fine-tunes PAK2 activity in response to various cellular signals.

What are the methodological considerations for studying PAK2 phosphorylation kinetics?

Studying the kinetics of PAK2 phosphorylation requires careful experimental design and consideration of several technical factors:

• Selection of kinetic approach: Traditional kinetic methods can be limited when studying fast activation reactions like PAK2 autophosphorylation. Researchers have developed specialized kinetic approaches to distinguish quantitatively between cis- and trans-pathways in autocatalytic reactions . These approaches involve mathematical modeling and careful time-course analyses.

• Temporal resolution: PAK2 autophosphorylation can occur rapidly, particularly in the trans-amplification phase. Experimental designs must incorporate appropriate time points to capture the initial cis phosphorylation and subsequent trans amplification events. Time courses of substrate reaction during PAK2 autoactivation provide valuable insights into these mechanisms .

• Substrate effects: The method developed for studying PAK2 autophosphorylation kinetics is particularly useful for assessing substrate effects on modification reactions. Different substrates can influence the kinetics of PAK2 activation, and these effects must be accounted for in experimental designs .

• Quantification techniques: Precise quantification of phosphorylation levels is essential. Western blotting with phospho-specific antibodies provides a semi-quantitative measure, while techniques like mass spectrometry can offer more precise quantification of phosphorylation stoichiometry at multiple sites simultaneously .

• Enzyme concentration considerations: The concentration of PAK2 in experimental systems can significantly impact the observed kinetics, particularly the balance between cis and trans phosphorylation mechanisms. Researchers should conduct experiments at multiple enzyme concentrations to fully characterize the kinetic parameters .

How do different PAK2 inhibitors affect Ser141 phosphorylation status?

Various inhibitors targeting PAK kinases demonstrate differential effects on PAK2 Ser141 phosphorylation, providing valuable tools for researchers studying PAK2 signaling pathways:

• IPA-3: This allosteric inhibitor shows context-dependent efficacy in reducing Ser141 phosphorylation. Studies have demonstrated that IPA-3 effectively reduces Ser141/144 phosphorylation in cells treated in suspension but shows limited efficacy in adherent monolayers . Furthermore, different molecular weight forms of PAK1/2 display varying sensitivities to IPA-3, with sensitivity decreasing with increasing apparent molecular weight .

• FRAX597: This ATP-competitive inhibitor has proven to be one of the most efficient inhibitors of PAK kinase activity, capable of inducing approximately 70% dephosphorylation of PAK1 Ser144 (analogous to PAK2 Ser141) at 4 μM concentration . Its efficacy appears more consistent across different cellular contexts compared to IPA-3.

• SFK inhibition by dasatinib: Somewhat surprisingly, inhibition of Src family kinases by dasatinib shows only moderate effects on Ser141/144 phosphorylation. The effect is comparable across all PAK isoforms and experimental conditions, suggesting that SFK contribution to PAK phosphorylation at this site is limited .

A comparative table of inhibitor effects on PAK2 Ser141 phosphorylation:

These different inhibitor profiles provide researchers with options for manipulating PAK2 activity in various experimental contexts, helping to dissect the specific roles of PAK2 Ser141 phosphorylation in different signaling pathways.

What are the technical challenges in distinguishing between PAK1, PAK2, and PAK3 phosphorylation?

Distinguishing between the phosphorylation states of different PAK family members presents several technical challenges for researchers:

• Sequence homology: PAK1, PAK2, and PAK3 share significant sequence homology, particularly around key phosphorylation sites. For example, Ser141 in PAK2 corresponds to Ser144 in PAK1, and these sites are surrounded by similar amino acid sequences . This homology makes it difficult to develop antibodies that exclusively recognize one phosphorylated PAK without cross-reactivity.

• Co-expression in biological systems: Many cell types and tissues express multiple PAK family members simultaneously, complicating the interpretation of experimental results. In certain cell lines like HeLa, PAK1 may be barely detectable while PAK2 is more abundant, making it challenging to quantify changes in PAK1 phosphorylation specifically .

• Molecular weight overlap: While the PAK isoforms have slightly different theoretical molecular weights, post-translational modifications can cause their apparent molecular weights on Western blots to overlap, making it difficult to definitively identify specific isoforms based solely on migration patterns .

• Antibody specificity: Many commercially available phospho-specific antibodies recognize multiple PAK family members when phosphorylated at their respective sites. For instance, antibodies raised against phosphorylated PAK1/2/3 may detect all three proteins when phosphorylated at their corresponding serine residues .

• Multiple phosphorylation forms: PAK proteins can exist in multiple phosphorylation states with different apparent molecular weights, further complicating interpretation. For example, multiple PAK1 bands with different sensitivities to inhibitors have been observed in HEK293T cells .

To address these challenges, researchers may need to employ multiple complementary approaches, including:

• Using siRNA or CRISPR to selectively knock down specific PAK isoforms

• Employing recombinant expression of tagged versions of specific PAK proteins

• Utilizing mass spectrometry to precisely identify phosphorylation sites and their associated proteins

• Combining multiple antibodies with different specificity profiles to build a comprehensive picture

How does cellular context influence PAK2 phosphorylation at Ser141?

Cellular context significantly impacts PAK2 phosphorylation at Ser141, with several factors affecting the phosphorylation status:

• Cellular adhesion state: Research has demonstrated striking differences in PAK inhibitor efficacy between cells in suspension versus adherent monolayers. For instance, the PAK inhibitor IPA-3 effectively reduces Ser141/144 phosphorylation in cells treated in suspension but shows limited efficacy in adherent monolayers . This suggests that cell-matrix interactions modulate PAK2 activation mechanisms and susceptibility to inhibition.

• Cell type-specific expression patterns: Different cell types exhibit varying levels of PAK isoforms, which can influence the observed phosphorylation patterns. For example, in HeLa cells, PAK1 is barely detectable, making it difficult to quantify changes in PAK1 phosphorylation, while PAK2 signals are more robust . This variation necessitates cell type-specific optimization of experimental protocols.

• Activation status of upstream regulators: The small GTPases CDC42 and RAC1 are primary upstream activators of PAK2. Their activation status, which depends on various cellular contexts and stimuli, directly influences PAK2 phosphorylation at Ser141. Binding of active CDC42 and RAC1 to PAK2 triggers conformational changes that facilitate autophosphorylation .

• Apoptotic stimuli: Under apoptotic conditions, particularly those involving DNA damage, PAK2 can be cleaved by caspases to generate PAK-2p34, an active fragment that translocates to the nucleus. This cleavage and the resulting fragments have distinct phosphorylation profiles and functions compared to the full-length protein .

• Growth factor signaling: Growth factors like EGF can influence PAK2 phosphorylation through activation of upstream signaling molecules. PAK2 plays an important role in EGF-induced cell proliferation through phosphorylation of JUN, indicating that growth factor context significantly impacts PAK2 phosphorylation status .

These contextual influences underscore the importance of carefully considering and controlling experimental conditions when studying PAK2 phosphorylation, as results obtained in one cellular context may not directly translate to others.

What methods can be used to validate antibody specificity for phospho-PAK2 (Ser141)?

Validating antibody specificity for phospho-PAK2 (Ser141) is crucial for ensuring reliable experimental results. Several complementary approaches can be employed:

• Phosphatase treatment: Treating cell lysates with phosphatases (e.g., lambda phosphatase) should eliminate the signal from phospho-specific antibodies while leaving total PAK2 detection unaffected. This serves as a direct confirmation that the antibody is detecting a phosphorylated epitope rather than total protein .

• Phosphorylation site mutants: Generating PAK2 constructs with serine-to-alanine mutations at position 141 (S141A) provides an excellent negative control. When expressed in cells, these mutants should not be detected by phospho-specific antibodies even under conditions that normally promote phosphorylation .

• Kinase inhibitor treatment: Treating cells with PAK inhibitors like FRAX597, which has been shown to reduce Ser141 phosphorylation by approximately 70%, should correspondingly reduce antibody signal if it is truly specific for the phosphorylated form .

• Peptide competition assays: Pre-incubating the antibody with excess phosphorylated peptide corresponding to the Ser141 region (e.g., Y-L-S(p)-F-T) should block antibody binding and eliminate signal. In contrast, pre-incubation with the non-phosphorylated version of the same peptide should have minimal effect on a truly phospho-specific antibody .

• Isoform-specific knockdown: Using siRNA or CRISPR to specifically reduce PAK2 expression can help confirm that the observed signal derives from PAK2 rather than other PAK family members. This is particularly important given the sequence homology between PAK1, PAK2, and PAK3 .

• Correlation with known activators: Treatment with established PAK2 activators, such as constitutively active forms of CDC42 or RAC1, should increase phosphorylation at Ser141. The antibody signal should correspondingly increase if it is specific for phospho-Ser141 .

• Mass spectrometry validation: For the most rigorous validation, immunoprecipitation followed by mass spectrometry can definitively identify the phosphorylation site being detected by the antibody, confirming both the identity of the protein (PAK2) and the specific phosphorylation site (Ser141) .

How does caspase-mediated cleavage of PAK2 affect Ser141 phosphorylation and its detection?

Caspase-mediated cleavage of PAK2 represents a critical regulatory mechanism with significant implications for Ser141 phosphorylation and its detection:

• Cleavage mechanism and products: During apoptosis, particularly in response to DNA damage, PAK2 undergoes caspase-mediated cleavage, generating an active p34 fragment called PAK-2p34 . This cleavage occurs downstream of the Ser141 phosphorylation site, potentially separating this site from the catalytic domain.

• Localization changes: While full-length PAK2 primarily functions in the cytoplasm, the cleaved PAK-2p34 fragment translocates to the nucleus where it promotes cellular apoptosis through the JNK signaling pathway . This compartmentalization may affect the detection of Ser141 phosphorylation in subcellular fractionation experiments.

• Functional conversion: Full-length phosphorylated PAK2 generally promotes cell survival and growth, contrasting sharply with the pro-apoptotic function of the cleaved PAK-2p34 fragment . This functional conversion may be associated with changes in the phosphorylation profile, including at Ser141.

• Detection challenges: When using Western blotting to detect phospho-PAK2 (Ser141), researchers must consider the molecular weight of the bands observed. The cleaved PAK-2p34 fragment would appear at a lower molecular weight compared to full-length PAK2 (approximately 34 kDa vs. 60 kDa) . If the Ser141 site remains on the p34 fragment, phospho-specific antibodies might detect both forms.

• Temporal dynamics: The time course of caspase activation, PAK2 cleavage, and changes in phosphorylation status may not be synchronized. Researchers investigating apoptotic pathways should conduct careful time-course experiments to capture these dynamic changes .

• Substrate specificity changes: Caspase-activated PAK2 has been shown to phosphorylate MKNK1 and reduce cellular translation, suggesting that cleavage may alter substrate specificity compared to full-length PAK2 . This could involve changes in the phosphorylation status of regulatory sites like Ser141.

For researchers studying PAK2 in apoptotic contexts, it is advisable to use both phospho-specific antibodies and total PAK2 antibodies that can detect both full-length and cleaved forms, enabling a comprehensive understanding of PAK2 regulation during apoptosis.

What is the recommended Western blotting protocol for optimal detection of phospho-PAK2 (Ser141)?

A comprehensive Western blotting protocol optimized for phospho-PAK2 (Ser141) detection should include the following key steps:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) to preserve phosphorylation status

  • Include protease inhibitors to prevent degradation of full-length PAK2

  • Maintain samples at 4°C throughout processing to minimize phosphatase activity

  • Determine protein concentration using a compatible assay (BCA or Bradford)

• Gel electrophoresis:

  • Use 8-10% SDS-PAGE gels for optimal resolution around the 60 kDa range where PAK2 migrates

  • Load 20-50 μg of total protein per lane, depending on PAK2 expression levels

  • Include molecular weight markers spanning 25-100 kDa range to accurately identify PAK2 bands

  • Consider including both phosphorylated and non-phosphorylated control samples

• Transfer conditions:

  • Transfer proteins to PVDF or nitrocellulose membranes

  • Use wet transfer systems for higher molecular weight proteins

  • Transfer at 100V for 1-2 hours or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block membranes in 5% BSA in TBST (not milk, which contains phosphatases)

  • Dilute primary phospho-PAK2 (Ser141) antibody 1:500 to 1:3000 in 5% BSA/TBST

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Wash extensively with TBST (3-5 times, 5-10 minutes each)

  • Incubate with appropriate HRP-conjugated secondary antibody (typically anti-rabbit at 1:5000 to 1:10000) for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • For weak signals, consider using high-sensitivity ECL substrates

  • Image using a digital imaging system with exposure times optimized for the signal intensity

• Controls and normalization:

  • Strip and reprobe membranes for total PAK2 to normalize phospho-signal

  • Include a loading control (e.g., GAPDH or β-actin)

  • Consider including a positive control (e.g., cells treated with CDC42/RAC1 activators)

• Troubleshooting common issues:

  • High background: Increase blocking time, use fresh blocking agent, increase wash stringency

  • No signal: Verify primary antibody concentration, check protein loading, confirm activation of PAK2

  • Multiple bands: Evaluate specificity with peptide competition, consider cross-reactivity with PAK1/PAK3

How can cell-based ELISA techniques be optimized for quantifying phospho-PAK2 (Ser141)?

Cell-based ELISA techniques offer an alternative approach for quantifying phospho-PAK2 (Ser141) directly in cultured cells. Based on the information from search result , here is an optimized protocol:

• Experimental design considerations:

  • Select appropriate cell lines with detectable PAK2 expression

  • Design experiments to include suitable positive controls (CDC42/RAC1 activators) and negative controls (PAK inhibitors like FRAX597)

  • Plan for multiple normalization methods to ensure robust quantification

• Cell preparation and fixation:

  • Seed cells at consistent density in 96-well plates

  • Apply treatments at appropriate time points

  • Fix cells with paraformaldehyde to preserve phosphorylation status

  • Permeabilize with detergent solution to allow antibody access to intracellular targets

• Primary antibody incubation:

  • Block non-specific binding sites

  • Apply anti-phospho-PAK2 (Ser141) antibody at optimized concentration

  • Incubate overnight at 4°C for maximum sensitivity

• Detection system:

  • Incubate with HRP-conjugated secondary antibody

  • Add colorimetric substrate (e.g., TMB) and measure absorbance using a plate reader

• Normalization approaches:

  • Anti-GAPDH antibody as an internal positive control for normalizing target absorbance values

  • Crystal Violet whole-cell staining to determine cell density and adjust for plating differences

  • Anti-PAK2 antibody for normalizing phospho-PAK2 signal to total PAK2 levels

• Validation and quality control:

  • Verify specificity using phosphatase treatments

  • Confirm dose-response relationships with known activators and inhibitors

  • Establish standard curves if semi-quantitative analysis is required

• Data analysis considerations:

  • Calculate the ratio of phospho-PAK2 to total PAK2 signals

  • Normalize to cell number using Crystal Violet data

  • Compare experimental conditions using appropriate statistical methods

This cell-based ELISA approach provides advantages for high-throughput screening of compounds affecting PAK2 phosphorylation and enables analysis of phosphorylation dynamics in intact cells without the need for cell lysis and Western blotting.