PRKACA Human, sf9

What is PRKACA and what is its molecular structure when expressed in Sf9 cells?

PRKACA (Protein Kinase cAMP-Activated Catalytic Subunit Alpha) is the main catalytic subunit of cAMP-dependent protein kinase (PKA), serving as the primary effector of cAMP signaling in human tissues. When expressed in Sf9 baculovirus cells, it typically consists of a single glycosylated polypeptide chain containing 578 amino acids (1-351 a.a. of the native protein) with a molecular mass of approximately 67kDa . On SDS-PAGE under reducing conditions, it migrates within the 50-70kDa range due to glycosylation variations . Commercial preparations commonly include a 227 amino acid GST tag at the N-terminus to facilitate purification and downstream applications .

What cellular functions does PRKACA regulate?

PRKACA is responsible for phosphorylating various proteins and substrates, regulating their activity across multiple cellular processes. As a member of the AGC kinase family, PRKACA contributes to:

• Glucose metabolism regulation

• Cell division control

• Contextual memory formation

• Cell adhesion mechanisms

• Cell transformation processes

• Cell cycle checkpoint regulation

• Cell volume control

Dysregulation of PRKACA through mutation or fusion events has been implicated in various pathologies, including adrenocortical tumors and specific forms of liver cancer .

What are the optimal storage conditions for maintaining PRKACA Human, sf9 activity?

For optimal preservation of PRKACA activity:

• Short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used

• Long-term storage: Store at -20°C or preferably at -70°C for periods exceeding one month

• Aliquoting recommendation: Divide into smaller quantities after centrifugation to minimize freeze-thaw cycles

• Carrier protein addition: For extended storage, adding a carrier protein (0.1% HSA or BSA) is recommended

• Avoid repeated freeze-thaw cycles to maintain protein integrity and enzymatic activity

Expected stability is approximately 1 year at -70°C from the shipment date when stored properly .

How should researchers validate the purity and activity of PRKACA Human, sf9 preparations?

Validation methods include:

• SDS-PAGE analysis: Should reveal a predominant band at 67kDa (or within the 50-70kDa range)

• Purity assessment: Commercial preparations typically provide >85-95% purity as determined by densitometry

• Activity assays: Standard kinase assays using known substrates compared against established activity parameters

• Western blotting: Using anti-PRKACA specific antibodies to confirm identity

• Functional testing: Evaluation of phosphorylation activity on well-characterized substrates

Researchers should always perform validation tests before beginning critical experiments, particularly when using a new lot of the protein.

What buffer systems optimize PRKACA activity in in vitro kinase assays?

The optimal buffer composition typically includes:

These components are reflected in the formulations used for commercial PRKACA preparations, which typically contain phosphate-buffered saline (pH 7.4) with 10% glycerol .

How can researchers differentiate between PRKACA and other related kinases like PKCα in experimental settings?

While PRKACA and PKCα (Protein Kinase C alpha) may show some overlap in experimental settings, they can be differentiated by:

• Activation mechanisms:

  • PRKACA is primarily activated by cAMP

  • PKCα is activated by calcium and diacylglycerol

• Substrate specificity:

  • Each kinase has distinct substrate preferences despite some overlap

  • Substrate selection can be optimized using published consensus motifs

• Inhibitor profiles:

  • Selective inhibitors for each kinase family exist and should be employed as controls

• Cellular function:

  • PKCα regulates cardiac contractility and Ca²⁺ handling in myocytes

  • PKCα has been assigned to chromosome region 17q22-q23.2, while PRKACA has a different chromosomal location

• Molecular weight differences:

  • PKCα shows a higher molecular weight (approximately 103kDa) compared to PRKACA

How is PRKACA involved in the development of fibrolamellar hepatocellular carcinoma?

PRKACA plays a crucial role in fibrolamellar hepatocellular carcinoma (FL-HCC) through a specific gene fusion event:

• Genetic mechanism: A somatic 400 Kb deletion on chromosome 19 creates a fusion between DNAJB1 (DnaJ heat shock protein family member B1) and PRKACA genes

• Disease context: FL-HCC predominantly affects children and young adults with no underlying liver disease

• Causal relationship: CRISPR/Cas9 studies creating this fusion in mice demonstrated that the DNAJB1-PRKACA gene fusion alone is sufficient to induce tumors resembling FL-HCC

• Therapeutic implications: This genetic evidence suggests strategies to block DNAJB1-PRKACA activity might be developed as therapeutics for this form of liver cancer

This research highlights the importance of PRKACA in oncogenic pathways and provides a rationale for developing experimental models using recombinant PRKACA to screen potential inhibitors.

What role does PRKACA play in adrenocortical pathologies?

PRKACA mutations have been implicated in various adrenocortical disorders:

• Adrenocortical tumors: Mutations of PRKACA have been identified in the pathogenesis of adrenocortical tumors

• ACTH-independent Cushing syndrome (AICS): PRKACA dysregulation contributes to this condition

• Bilateral adrenocortical hyperplasias (BAH): PRKACA mutations are associated with this condition

• Regulatory context: PRKACA functions as part of the PKA holoenzyme, where its activity is normally regulated by regulatory subunits like PRKAR1A. Inactivating mutations of PRKAR1A are already known to cause Carney complex and primary pigmented nodular adrenocortical disease (PPNAD)

These connections make PRKACA Human, sf9 a valuable research tool for investigating the molecular mechanisms underlying these endocrine disorders.

What strategies can optimize experiments comparing wild-type PRKACA with disease-associated mutants?

When comparing wild-type PRKACA with mutant variants:

• Expression systems:

  • Maintain consistent expression conditions between wild-type and mutant proteins

  • Use the same tag system (e.g., GST) and purification protocol for both proteins

• Activity normalization:

  • Standardize protein concentrations and active site titrations

  • Conduct enzyme kinetics (Km and Vmax determination) under identical conditions

• Control experiments:

  • Include kinase-dead mutants as negative controls

  • Use specific inhibitors to validate that observed phosphorylation is PRKACA-dependent

• Substrate selection:

  • Test multiple substrates to detect changes in specificity

  • Include physiologically relevant substrates alongside standard kinase assay substrates

• Structural analysis:

  • Consider how mutations might affect protein folding, stability, or substrate access

  • When possible, complement activity assays with structural studies

How does the GST tag potentially influence PRKACA activity and experimental design?

The 227 amino acid GST tag commonly used with PRKACA Human, sf9 preparations may influence:

• Protein solubility and stability:

  • GST typically enhances solubility, potentially improving yield and handling properties

  • May provide additional stability during storage and experimental manipulations

• Enzymatic parameters:

  • The large tag (approximately 26 kDa) may subtly alter enzyme kinetics

  • Could potentially affect substrate accessibility or binding

• Experimental considerations:

  • For critical structural or interaction studies, consider tag removal via protease cleavage

  • In kinase assays, include appropriate controls that account for potential tag effects

  • When comparing results across studies, note whether tagged or untagged protein was used

• Protein interactions:

  • The tag might interfere with or introduce artificial protein-protein interactions

  • Consider this especially when studying PRKACA in complex with regulatory proteins

What are common challenges when working with PRKACA Human, sf9 and how can they be addressed?

How can researchers design appropriate controls for PRKACA activity assays?

Comprehensive controls for PRKACA activity assays should include:

• Positive controls:

  • Commercial active PRKACA with known specific activity

  • Well-characterized substrate with established phosphorylation kinetics

• Negative controls:

  • Heat-inactivated enzyme preparation (95°C for 5 minutes)

  • Kinase-dead mutant (e.g., K72R mutation in the ATP-binding site)

  • Reaction mixture without enzyme

• Specificity controls:

  • Specific PRKACA inhibitors to confirm that observed activity is PRKACA-dependent

  • Non-phosphorylatable substrate mutants (S/T→A mutations)

• System validation:

  • Phospho-specific antibody controls (if using antibody-based detection methods)

  • Standard curves with known amounts of phosphorylated substrate

These controls ensure experimental rigor and help troubleshoot when unexpected results occur.