Recombinant Callithrix jacchus Phosphatidylinositol 4-kinase beta (PI4KB), partial

What is Phosphatidylinositol 4-kinase beta (PI4KB) and what is its functional significance?

Phosphatidylinositol (PI) 4-kinases catalyze the synthesis of PI 4-phosphate, which serves as an important intermediate for the synthesis of membrane polyphosphoinositides that regulate multiple cellular functions. PI4KB specifically is a 110-kDa enzyme that can be inhibited by wortmannin concentrations exceeding 0.1 micromolar . The enzyme plays a critical role in phospholipid metabolism and membrane signaling pathways, making it a significant target for research in cellular regulation mechanisms.

Why is studying PI4KB specifically in Callithrix jacchus (common marmoset) valuable for research?

Callithrix jacchus represents an important non-human primate model in biomedical research. The marmoset genome contains unique evolutionary features, as evidenced by the identification of novel genomic elements such as the Platy-1 repeat family . Studying species-specific variations in enzymes like PI4KB provides valuable insights into evolutionary conservation and divergence of critical cellular signaling pathways in primates, potentially revealing unique adaptations in New World monkeys compared to other primates.

What does "partial" indicate in the context of recombinant Callithrix jacchus PI4KB?

The term "partial" likely refers to a recombinant protein that contains only a portion of the full-length PI4KB sequence, potentially focusing on the catalytic domain. Research indicates that the C-terminal catalytic domain of PI4KB can retain functionality even when isolated, as demonstrated by its ability to maintain autophosphorylation activity . Partial recombinant proteins are often used to study specific functional domains without interference from regulatory regions.

What expression systems are most effective for producing active recombinant PI4KB?

Based on established methodologies, E. coli expression systems using glutathione S-transferase (GST) fusion proteins have proven effective for recombinant PI4KB production. This approach has yielded biologically active protein that phosphorylates PI in its 4-position with wortmannin sensitivity and kinetic parameters identical to those of purified bovine brain PI4KB . For Callithrix jacchus PI4KB specifically, researchers should optimize codon usage for bacterial expression and determine appropriate induction conditions.

How can the enzymatic activity of recombinant Callithrix jacchus PI4KB be reliably measured?

A systematic approach to measuring PI4KB activity includes:

• Preparation of phosphatidylinositol substrates in appropriate micelles or vesicles

• Incubation with purified recombinant PI4KB and [γ-32P]ATP

• Extraction and analysis of lipids via thin-layer chromatography or HPLC to detect PI 4-phosphate formation

• Assessment in the presence and absence of inhibitors like wortmannin and LY 294002

• Additional measurement of autophosphorylation by incubating the enzyme with [γ-32P]ATP in the presence of Mn2+ ions

What purification strategy yields optimal results for recombinant Callithrix jacchus PI4KB?

A recommended purification protocol based on established methodologies involves:

What is known about the regulatory mechanisms controlling PI4KB activity?

Research on PI4KB has revealed several key regulatory mechanisms:

• Autophosphorylation serves as a primary regulatory mechanism that inhibits subsequent lipid kinase activity

• This inhibition can be reversed by protein phosphatases PP1 and PP2A(1), suggesting a dynamic regulatory system

• Autophosphorylation is enhanced by Mn2+ ions and inhibited by wortmannin and LY 294002

• The autophosphorylation site resides within the C-terminal catalytic domain of the protein

• In vivo phosphorylation appears to involve both autophosphorylation and phosphorylation by other kinases

How does the autophosphorylation of PI4KB affect its enzymatic function?

Autophosphorylation appears to serve as a negative feedback mechanism for PI4KB activity. Research demonstrates that autophosphorylation inhibits subsequent lipid kinase activity, and this inhibition can be reversed upon dephosphorylation by protein phosphatases (PP1 and PP2A(1)) . The autophosphorylation site is maintained in position by intramolecular interactions rather than intermolecular ones, as the recombinant protein was unable to transphosphorylate but could still autophosphorylate .

What experimental approaches can be used to investigate the structural determinants of PI4KB autophosphorylation?

Several methodological approaches can be employed:

• Domain deletion and mutation analysis to identify specific residues involved in autophosphorylation

• Manipulation of reaction conditions to enhance (Mn2+ addition) or inhibit (wortmannin treatment) autophosphorylation

• Phosphatase treatment experiments to reverse autophosphorylation and restore lipid kinase activity

• Mass spectrometry analysis of phosphorylated protein to identify specific phosphorylation sites

• Crystallographic studies comparing phosphorylated and non-phosphorylated forms

How might PI4KB from Callithrix jacchus compare to that of other primate species?

While specific comparative data for Callithrix jacchus PI4KB is not directly provided in the search results, several inferences can be made:

• As a New World monkey, Callithrix jacchus belongs to a lineage that diverged from the catarrhine lineage (Old World monkeys and apes) around the time that the Platy-1 repeat family arose

• The catalytic domain of PI4KB is likely highly conserved across primate species due to functional constraints

• Regulatory regions may show more evolutionary divergence, potentially reflecting species-specific adaptations

• Comparative analysis of PI4KB across the primate phylogeny could reveal selective pressures acting on different protein domains

What unique genomic features of Callithrix jacchus might influence PI4KB expression or function?

The Callithrix jacchus genome contains several distinctive features that could potentially impact gene expression patterns:

• The presence of the novel Platy-1 repeat family, which is specific to New World monkeys and contains 2268 full-length elements across 62 subfamilies in the common marmoset genome

• Variation in GC content across chromosomes, which might influence gene expression patterns

• Unique distribution of repetitive elements that could affect chromatin structure or gene regulation

• Species-specific promoter elements that might confer unique expression patterns to genes including PI4KB

How can recombinant Callithrix jacchus PI4KB be utilized in drug development research?

Recombinant Callithrix jacchus PI4KB could serve several functions in drug development:

• As a target for screening potential inhibitors, similar to how wortmannin and LY 294002 have been shown to inhibit PI4KB

• For comparative studies evaluating inhibitor efficacy across species, essential for validating non-human primate models

• To identify novel regulatory mechanisms that could be targeted pharmaceutically

• For developing assays to evaluate the effect of compounds on phosphoinositide metabolism in a primate model system

What advanced techniques can be employed to study the interaction between PI4KB and cellular membranes?

Several sophisticated methodological approaches can address this question:

• Liposome binding assays with defined lipid compositions to determine membrane association requirements

• Fluorescently tagged PI4KB constructs for live-cell imaging of membrane association dynamics

• FRET-based approaches to monitor protein-lipid interactions in real-time

• Surface plasmon resonance to quantify binding kinetics to membrane mimetics

• Electron microscopy techniques to visualize PI4KB association with membrane structures

What are common challenges in expressing and purifying active recombinant Callithrix jacchus PI4KB, and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant kinases:

How can researchers differentiate between autophosphorylation and substrate phosphorylation when measuring PI4KB activity?

To distinguish between these activities:

• Perform kinase assays with and without PI substrate to isolate autophosphorylation activity

• Use a catalytically inactive mutant to determine if phosphorylation still occurs in vivo (indicating external kinases)

• Employ protein phosphatases to reversibly inhibit autophosphorylation and measure effects on lipid kinase activity

• Utilize phospho-specific antibodies if the autophosphorylation sites are known

• Employ mass spectrometry to identify specific phosphorylation sites on the protein versus substrate

Through these methodological approaches, researchers can effectively isolate and characterize the distinct phosphorylation activities of PI4KB from Callithrix jacchus, advancing our understanding of this enzyme's complex regulatory mechanisms.