PSKH2 Antibody

What is PSKH2 and why is it classified as a pseudokinase?

PSKH2 belongs to the protein kinase superfamily and CAMK Ser/Thr protein kinase family. In primates, PSKH2 is classified as a pseudokinase because it contains an HRN motif instead of the canonical HRD motif in the activation loop, with asparagine replacing the catalytic aspartate residue (N183) . This substitution significantly compromises catalytic activity, as the HRD-Asp functions as a putative catalytic base critical for phosphoryl transfer . Biochemical analyses confirm that human PSKH2 lacks detectable protein phosphotransferase activity, even when common non-specific substrates like myelin basic protein, α-casein, and histone proteins are used in in vitro kinase assays .

Interestingly, PSKH2 in most non-primate higher chordates retains a conventional HRD motif, suggesting that the Asn-pseudokinase switch is a relatively recent evolutionary event . Despite this evolutionary divergence, both forms of PSKH2 (with HRD or HRN motifs) lack detectable autophosphorylation activity, indicating that additional factors beyond the canonical motif may influence its functionality .

What structural features characterize PSKH2?

AlphaFold 2 (AF2) structural predictions reveal that PSKH2 adopts a typical bi-lobal kinase domain conformation (residues 63-320) with medium-to-high confidence (pLDDT > 70) . The kinase domain is flanked by largely disordered N-terminal (residues 1-35) and C-terminal (residues 344-385) regions .

Key structural elements include:

• Two conserved prolines (Pro36 and Pro57) that initiate alpha-helical structures NTH1 and NTH2 in the N-terminal region

• A conserved proline (Pro346) in the C-terminal region that shortens the first C-terminal helix compared to PSKH1

• A putative N-terminal myristoylation site at Gly2, which may facilitate membrane targeting

• Retention of all amino acid residues typically required to coordinate ATP binding, suggesting preserved nucleotide-binding capacity despite lacking catalytic activity

BPPS (Bayesian Partitioning with Pattern Selection) analysis has identified a subset of co-conserved residues (His291, Leu303, His307, and Asp316) that distinguish PSKH2 from closely related PSKH1 . These residues map to the substrate-binding C-lobe of the PSKH2 kinase domain model, presumably conferring specificity in protein-protein interactions .

How does the N-terminal domain affect PSKH2 expression and stability?

The N-terminal domain plays a critical role in PSKH2 expression and stability. Experimental evidence shows:

• Removal of the N-terminal region is poorly tolerated and results in loss of PSKH2 expression as demonstrated by immunoblotting

• This loss of expression cannot be rescued by the proteasome inhibitor MG132, suggesting the N-terminal dependency is not related to proteasomal degradation

• Incremental truncation of the N-terminus results in loss of PSKH2 expression following deletion of the first ~25 amino acids, while smaller truncations have minimal impact

The critical nature of the N-terminus may relate to the predicted alpha-helical structural element (NTH1, between residues 36-53) that was eliminated in truncation constructs . Interestingly, mutation of the predicted putative sites of myristoylation (Gly2) and/or palmitoylation (Cys3) does not cause observable loss of PSKH2 expression, indicating that these post-translational modifications are not the critical determinants for protein stability observed in N-terminal truncated forms .

What are the optimal methods for detecting endogenous PSKH2 in human samples?

Detection of endogenous PSKH2 presents significant challenges due to its generally "extremely low" or "absent" expression in most well-studied laboratory cell lines, including HEK-293 . For optimal detection, consider the following methodological approach:

Table 1: Recommended Applications and Dilutions for PSKH2 Antibodies

Target Tissues/Cells for Detection:

• HepG2 cells (human liver hepatocellular carcinoma)

• Human kidney tissue

• Human brain tissue

• Human heart tissue

• Human testis tissue

For enhanced specificity, blocking peptides that bind specifically to the target antibody are recommended . These peptides resemble the epitope for which the antibody is specific, thereby preventing undesirable binding. Pre-adsorption with blocking peptide should be performed by adding the peptide to the diluted primary antibody in a 10:1 molar ratio (peptide:antibody) and incubating at 4°C overnight or at room temperature for 2 hours .

How can researchers differentiate between PSKH1 and PSKH2 in experimental systems?

Distinguishing between the closely related PSKH1 and PSKH2 proteins requires careful experimental design. The following strategies can be employed:

Structural and Functional Differences:

• Catalytic activity: PSKH1 has kinase activity, while PSKH2 in primates lacks phosphotransferase activity due to the HRN motif

• Ca²⁺/Calmodulin regulation: PSKH1 activity is modulated by Ca²⁺/Calmodulin binding, while PSKH2 lacks this regulation

• Subcellular localization: PSKH1 contains a validated Golgi-targeting motif that is conspicuously absent from PSKH2

Methodological Approaches:

• Use antibodies targeting unique epitopes in the N- or C-terminal regions where the sequences diverge significantly

• Design PCR primers or probes targeting non-conserved regions for transcript analysis

• Exploit the different molecular weights (PSKH1: ~50 kDa; PSKH2: ~35 kDa) in western blotting

• Perform co-localization studies with organelle markers (Golgi for PSKH1, mitochondria for PSKH2)

• Use kinase activity assays to differentiate between catalytically active PSKH1 and inactive PSKH2

For species considerations, note that PSKH2 is absent from mouse and rat genomes, which prohibits standardized genetic knock-out approaches in these common model organisms . This makes rodent models suitable only for PSKH1 research, providing a natural system for studying PSKH1-specific functions.

What experimental approaches are most effective for studying PSKH2's role in mitochondrial networks?

Mass spectrometry-based proteomics has confirmed that human PSKH2 is part of a cellular mitochondrial protein network . To investigate this association, consider the following comprehensive approach:

• Subcellular Localization Studies:

  • Perform subcellular fractionation to isolate membrane-rich fractions containing mitochondrial proteins

  • Use immunofluorescence with co-staining for mitochondrial markers

  • Apply super-resolution microscopy for precise localization within mitochondrial structures

• N-terminal Domain Analysis:

  • Investigate the role of the myristoylated N-terminus in targeting PSKH2 to membrane-rich cellular fractions

  • Generate constructs with mutations in the myristoylation site (Gly2) and assess impact on mitochondrial localization

  • Create fusion proteins with fluorescent tags that preserve the critical N-terminal domain

• Protein-Protein Interaction Analysis:

  • Perform co-immunoprecipitation followed by mass spectrometry to identify mitochondrial binding partners

  • Use proximity labeling approaches (BioID, APEX2) to identify proteins in close proximity to PSKH2

  • Validate key interactions using reciprocal co-IP or FRET/BRET techniques

• Functional Impact Assessment:

  • Modulate PSKH2 expression and measure effects on mitochondrial membrane potential

  • Assess changes in mitochondrial morphology, dynamics, and quality control

  • Investigate potential roles in mitochondrial protein import or translation

• Disease-Relevant Contexts:

  • Study PSKH2 in cancer models, given its reported elevation in 5-10% of cancer cohorts

  • Investigate potential synthetic lethality with RAS oncogene, consistent with a role in proliferative signaling

  • Examine the impact of cancer-associated PSKH2 mutations on mitochondrial function

When designing these experiments, consider that PSKH2 expression is regulated through client-status within the HSP90/Cdc37 molecular chaperone system , which may influence its stability and localization in mitochondrial networks.

How can researchers analyze PSKH2 post-translational modifications?

Analysis of PSKH2 post-translational modifications (PTMs) requires specialized approaches due to its low expression levels and challenging purification. Based on current research, the following strategy is recommended:

• Protein Isolation Strategy:

  • Affinity purify PSKH2 using a cleavable tandem C-terminal Strep tag (to maintain an intact N-terminus with modifiable sites)

  • Use 3C-mediated cleavage for tag removal to minimize interference with native PTMs

  • Confirm purified protein identity using a validated PSKH2 antibody

• Mass Spectrometry Workflow:

  • Digest purified PSKH2 with multiple proteolytic enzymes (trypsin, chymotrypsin, elastase) to maximize sequence coverage (~63% coverage was achieved using this approach)

  • Perform phosphopeptide-enrichment prior to LC-MS/MS analysis

  • Analyze samples using high-resolution mass spectrometry with both CID and ETD fragmentation modes

• Key Modification Sites to Monitor:

  • Tyr228 phosphorylation (consistently observed in previous studies)

  • N-terminal myristoylation (Gly2) and palmitoylation (Cys3) sites

  • Six phosphorylation sites catalogued in PhosphoSite Plus, although these have generally only been identified in single high-throughput studies

  • Potential deamidation of Asn183 (which might theoretically reinstate catalytic function by converting to Asp)

• Validation Methods:

  • Use phospho-specific antibodies against identified sites for western blotting

  • Confirm PTMs using site-directed mutagenesis of modified residues

  • Apply metabolic labeling with bioorthogonal reporters for lipid modifications

  • Assess functional consequences of modifications on localization, stability, and interactions

What are the best methods to study the HSP90/Cdc37 chaperone system's regulation of PSKH2?

The interaction between PSKH2 and the HSP90/Cdc37 molecular chaperone system represents a key regulatory mechanism for this pseudokinase. Mass spectrometry-based proteomics has confirmed that human PSKH2 is regulated through client-status within this chaperone system, with HSP90 interactions mediated through binding to the PSKH2 C-terminal tail . To study this relationship effectively:

• C-terminal Domain Analysis:

  • Generate C-terminal truncation constructs to map the specific regions mediating HSP90 interaction

  • Create point mutations in the C-terminal tail to identify critical residues

  • Compare wild-type and C-terminal mutant PSKH2 for stability and localization differences

• Pharmacological Approach:

  • Treat cells expressing PSKH2 with HSP90 inhibitors (geldanamycin, 17-AAG, radicicol)

  • Monitor PSKH2 levels, stability, and localization following chaperone inhibition

  • Perform dose-response and time-course studies to characterize dependency

• Protein-Protein Interaction Analysis:

  • Perform co-immunoprecipitation of PSKH2 followed by western blotting for HSP90 and Cdc37

  • Use proximity ligation assay (PLA) to detect and quantify interactions in situ

  • Apply FRET or BiFC approaches to monitor interaction dynamics in living cells

• Functional Consequences:

  • Investigate how disruption of HSP90 interaction affects PSKH2 association with mitochondrial networks

  • Assess impact on any identified PSKH2-dependent cellular processes

  • Study whether HSP90/Cdc37 regulation is altered in disease states where PSKH2 is implicated

This regulatory system is particularly significant because the C-terminal tail of PSKH2 may act as both a cis and trans regulatory element, driving outputs linked to the PSKH2 pseudokinase domain that are important for functional signaling . In PSKH1, this region serves as a Ca²⁺/CaM docking site, suggesting a potential parallel regulatory mechanism for PSKH2 .