Recombinant Casein kinase I (CK1)

What is Casein Kinase I (CK1) and what are its main functions?

Casein Kinase I (CK1) is a widely expressed Ser/Thr kinase family found in eukaryotic organisms that plays crucial roles in various cellular processes. CK1 was initially characterized by its preferential phosphorylation of acidic proteins such as caseins as substrates . Despite this operational definition, CK1 has been shown to phosphorylate a diverse array of proteins in vivo and in vitro .

The CK1 family is involved in regulating several fundamental cellular processes including:

• Wnt signaling pathway, where it phosphorylates β-catenin (CTNNB1) at Ser-45

• Circadian rhythm regulation through potential phosphorylation of PER1 and PER2 proteins

• Cytoskeletal organization, particularly in keratin cytoskeleton disassembly and epithelial cell migration

• Cell cycle progression and mitotic chromosome segregation

• DNA repair processes, with certain CK1 family members showing conserved catalytic faces associated with DNA repair regulation

• mTORC1 and mTORC2 signaling in response to nutrients by mediating DEPTOR phosphorylation

• Inflammatory response regulation through potential inhibition of NLRP3 inflammasome assembly

These diverse functions make CK1 a critical target for research across multiple fields from basic cell biology to disease-focused studies.

What are the different isoforms of CK1 and how do they differ structurally?

The CK1 family consists of several isoforms that share significant sequence homology but differ in size, tissue distribution, and specific functions. The main mammalian isoforms include:

All CK1 isoforms share a highly conserved catalytic domain but differ primarily in their C-terminal regions, which often contain regulatory elements including autoinhibitory domains . The sequence conservation is especially high in regions associated with ATP binding and catalysis, while more divergence is seen in substrate recognition regions and regulatory domains .

What is the three-dimensional structure of CK1 and how does it relate to function?

The three-dimensional structure of mammalian CK1δ's catalytic region has been solved by X-ray crystallography to a resolution of 2.3 Å . Like other protein kinases, the catalytic domain of CK1 consists of two lobes with a cleft between them that serves as the ATP-binding site .

Key structural features include:

• N-terminal lobe: Undergoes rotation upon ATP binding, similar but not identical to the domain motion observed in cAMP-dependent protein kinase

• C-terminal lobe: Contains catalytic residues and substrate recognition elements

• ATP-binding cleft: Located between the two lobes

• Anion binding site: Identified using tungstate derivatives, this site may contribute to the unique substrate specificity of CK1

• Autoinhibitory region: In full-length CK1δ, the C-terminal region functions as an autoinhibitory domain, though crystallographic studies have found this region to be disordered in crystal structures

Structural comparison between mammalian CK1δ and its yeast homolog Cki1 revealed a conserved catalytic face that is especially preserved among CK1 family members associated with DNA repair regulation . This conservation suggests evolutionary pressure to maintain specific structural features related to this function.

A notable feature observed in crystallographic studies is the potential formation of dimers through conserved intermolecular contacts, which might inhibit kinase activity . This could represent an additional regulatory mechanism for CK1 function.

Why does recombinant CK1 undergo autoinactivation in E. coli and how can this be prevented?

Recombinant CK1 undergoes autoinactivation via autophosphorylation when expressed in standard Escherichia coli expression systems . This represents a significant challenge for researchers attempting to produce active enzyme for in vitro studies. The autophosphorylation occurs due to the intrinsic kinase activity of CK1 during expression, resulting in a phosphorylated and consequently inactivated form of the enzyme .

The primary mechanism behind this issue is:

• During expression in E. coli, CK1 catalyzes the transfer of phosphate groups from ATP to specific residues on itself

• These autophosphorylation events occur in regions that regulate kinase activity

• The resulting phosphorylated CK1 has significantly reduced catalytic activity compared to the unphosphorylated form

To circumvent this problem, researchers have developed several strategies:

• Co-expression with phosphatases: A novel protein expression system using E. coli strain BL21(DE3)pλPP has been established, which constitutively expresses λ protein phosphatase (λPPase) . This approach allows:

  • Prevention of accumulation of phosphorylated CK1 during expression

  • Production of recombinant CK1 isoforms (α, δ, and ε) in unphosphorylated forms

  • Markedly higher enzymatic activity compared to CK1 prepared using conventional BL21(DE3)

• In vitro dephosphorylation: Conventional methods involve post-expression treatment with λPPase, but this approach is more laborious and less efficient than the co-expression system .

• Truncation mutants: Expressing only the catalytic domain without the C-terminal autoinhibitory region can produce active kinase, though this may alter some regulatory properties .

The BL21(DE3)pλPP expression system represents a significant methodological advancement that may also be applicable to other kinases that are difficult to prepare in active form due to phosphorylation during expression in E. coli .

What are the key methodological considerations for crystallizing recombinant CK1?

Crystallization of recombinant CK1 for structural studies involves several critical methodological considerations, as evidenced by successful crystallographic studies:

• Protein construction decisions:

  • Truncation mutants lacking the C-terminal autoinhibitory region have been successfully crystallized

  • Full-length CK1δ containing both catalytic and autoinhibitory domains has been crystallized, but electron density for the inhibitory domain was not interpretable, suggesting disorder in this region

• Expression and purification strategy:

  • Expression in E. coli systems, with careful consideration of phosphorylation status

  • Purification protocols must maintain protein stability and homogeneity

• Crystallization conditions:

  • CK1δ crystals have been obtained that belong to space group C2221

  • Resolution of 2.3-2.4 Å has been achieved for CK1δ structures

• Structure solution approaches:

  • Molecular replacement techniques using related structures have been successful

  • Tungstate derivatives have been used to confirm molecular replacement solutions and identify anion binding sites

• Challenges to consider:

  • The C-terminal autoinhibitory domain appears disordered in crystal structures, complicating full structural characterization

  • Intermolecular contacts observed in crystals suggest potential dimer formation, which may reflect physiologically relevant interactions or crystallization artifacts

These considerations highlight the technical challenges in obtaining high-quality structural data for CK1 and the importance of carefully designed constructs for crystallographic studies.

How does substrate specificity of CK1 relate to its structure?

The substrate specificity of CK1 is directly related to its three-dimensional structure and involves several distinctive features:

• Anion binding site: Crystallographic studies using tungstate derivatives identified a specific anion binding site that likely contributes to the unique substrate specificity of CK1 . This site may recognize phosphorylated residues in substrates, explaining the preference of CK1 for substrates with prior phosphorylation.

• Substrate recognition elements: The CK1 structure contains specific residues involved in substrate recognition that differ from those in other protein kinases . These residues create a unique catalytic face that determines which proteins can serve as substrates.

• Conservation patterns: Alignment of different CK1 isoforms reveals that while ATP-binding residues are highly conserved, substrate recognition residues show more variation between isoforms . This variation likely contributes to the different substrate preferences of CK1 family members.

• Preference for acidic substrates: CK1 preferentially phosphorylates substrates with acidic residues, such as caseins . This preference is reflected in the structure of the substrate-binding region, which contains positively charged residues that interact with acidic residues in substrates.

• Kinetic behavior with different substrates: The interaction of CK1 with substrates can be modulated by regulatory factors. For instance, heparin inhibits recombinant CK1δ when phosvitin is the substrate (half-maximal inhibition at 11.5 μg/ml) but activates it 4-5 fold when a peptide substrate is used (half-maximal activation at 9.5 μg/ml) . This differential effect suggests conformational changes in CK1 that affect substrate recognition.

What expression systems are optimal for producing highly active recombinant CK1?

The optimal expression system for producing highly active recombinant CK1 depends on several factors, with the bacterial system using phosphatase co-expression showing particular promise:

• E. coli BL21(DE3)pλPP system:

  • This specialized strain constitutively expresses λ protein phosphatase (λPPase)

  • The system effectively prevents autophosphorylation-mediated inactivation during expression

  • CK1 isoforms (α, δ, and ε) produced using this system show markedly higher activity than those prepared using conventional BL21(DE3)

  • This approach eliminates the need for troublesome in vitro λPPase treatment steps

• Conventional E. coli BL21(DE3) with modifications:

  • Expression of truncated forms lacking the C-terminal autoinhibitory domain

  • Post-purification treatment with phosphatases to remove inhibitory phosphorylation

  • Optimization of induction conditions to minimize autophosphorylation

• Key factors affecting expression efficiency:

  • Temperature: Lower expression temperatures can reduce autophosphorylation rates

  • Induction duration: Shorter induction periods may limit the extent of autophosphorylation

  • Media composition: Nutrient availability affects expression levels and protein folding

  • Codon optimization: Adapting the CK1 coding sequence to E. coli codon usage can improve expression

• Purification considerations:

  • Affinity tags selection: Appropriate tags can facilitate purification without interfering with activity

  • Removal of C-terminal degradation products: Expression of CK1δ in E. coli results in not only the full-length 55-kDa protein but also C-terminal degradation products of 50 and 42 kDa

  • Verification of phosphorylation status: Ensuring the recombinant CK1 is in the desired phosphorylation state

The BL21(DE3)pλPP system represents the most efficient approach currently available for producing highly active CK1 and may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells .

How can the activity of recombinant CK1 be accurately measured?

Accurate measurement of recombinant CK1 activity is essential for characterizing the enzyme and evaluating the success of expression and purification protocols. Several complementary approaches can be used:

• Standard kinase activity assays:

  • Casein phosphorylation: CK1 was initially defined by its ability to phosphorylate casein

  • Phosvitin phosphorylation: Another acidic protein substrate used to measure CK1 activity

  • Synthetic peptide substrates: CK1δ can phosphorylate specific peptides such as DDDDVASLPGLRRR

• Activity modulation tests:

  • Inhibition by CKI-7: Recombinant CK1δ is inhibited by the specific CK1 inhibitor CKI-7, with half-maximal inhibition at 12 μM

  • Heparin response: Depending on the substrate used, heparin can either inhibit or activate CK1, providing information about the enzyme's conformation and regulation

• Phosphorylation state analysis:

  • Western blotting with phospho-specific antibodies

  • Mass spectrometry to identify and quantify phosphorylation sites

  • Mobility shift assays to detect changes in electrophoretic mobility due to phosphorylation

• Comparison metrics:

  • Relative activity: CK1 prepared using BL21(DE3)pλPP shows markedly higher activity than preparations from conventional BL21(DE3)

  • Activity after phosphatase treatment: Comparing activity before and after in vitro λPPase treatment provides information about the degree of inhibitory phosphorylation

• Substrate specificity profiling:

  • Testing activity against multiple substrates provides information about substrate recognition and enzyme conformation

  • Altered specificity may indicate structural changes or regulatory modifications

These methods provide complementary information about CK1 activity and can be selected based on the specific research question and available resources.

What genetic engineering approaches are used for CK1 studies?

Several genetic engineering approaches have been developed to study CK1 function, structure, and regulation:

• Gene knockout and mutation strategies:

  • CSNK1G3 gene knockout protocols have been established for human cells

  • C-terminally truncated CSNK1G3 mutants can be identified using lysenin-resistance analysis by MTT assay

  • Retroviral vector systems enable cloning and expression of CSNK1G1-3 cDNAs

• Domain modification approaches:

  • Truncation mutants: Removal of the C-terminal autoinhibitory region (111 amino acids in CK1δ) alters regulatory properties such as heparin response

  • Catalytic domain isolation: Expression of only the catalytic domain facilitates structural studies

  • Full-length constructs: Including the autoinhibitory domain allows study of regulatory mechanisms

• Expression constructs for recombinant production:

  • cDNA cloning: Full-length CK1δ cDNA contains an open reading frame of 1284 nucleotides encoding a 428-amino acid polypeptide

  • Bacterial expression vectors: Optimized for high-level expression in E. coli

  • Mammalian expression vectors: For studying CK1 in cellular contexts

• Site-directed mutagenesis applications:

  • Mutation of residues involved in ATP binding allows study of catalytic mechanisms

  • Alteration of substrate recognition residues provides insights into specificity determinants

  • Modification of potential phosphorylation sites helps understand autoregulation

• Tissue-specific expression analysis:

  • Northern blotting reveals tissue-specific expression patterns, with CK1δ showing three hybridizing species (3.5-4.1, 2.2, and 1.9 kb), with the 1.9- and 2.2-kb species predominantly found in testis

  • 3'-untranslated region probes can distinguish between specific transcripts

These genetic engineering approaches provide powerful tools for understanding CK1 biology, developing improved recombinant production systems, and potentially identifying therapeutic targets.

How can problems with low activity of recombinant CK1 be addressed?

Low activity of recombinant CK1 is a common challenge that can be addressed through several targeted approaches:

• Identifying the cause of low activity:

  • Autophosphorylation: Recombinant CK1 undergoes autoinactivation via autophosphorylation in E. coli cells

  • Improper folding: Non-native conformations may result in reduced activity

  • C-terminal degradation: Expression in E. coli can produce C-terminal degradation products with potentially altered activity

  • Inhibitory factors: Components of the expression or purification system may inhibit CK1 activity

• Expression system optimization:

  • Use of BL21(DE3)pλPP strain: This E. coli strain constitutively expresses λ protein phosphatase, preventing autophosphorylation during expression

  • Temperature reduction: Lower expression temperatures can improve protein folding

  • Induction conditions: Optimizing IPTG concentration and induction duration

• Post-expression treatments:

  • In vitro λPPase treatment: Dephosphorylation can recover activity of autophosphorylated CK1

  • Proper storage conditions: Using appropriate buffers and additives to maintain stability

  • Addition of activators: Certain substrates or conditions may enhance activity

• Construct design considerations:

  • Expression of truncated forms: Removing the C-terminal autoinhibitory domain can increase basal activity

  • Codon optimization: Adapting the coding sequence to the expression host

  • Fusion tags: Selection of appropriate fusion partners that don't interfere with activity

• Activity assay selection:

  • Different substrates: CK1 activity varies depending on the substrate (casein, phosvitin, peptides)

  • Effect of regulators: Heparin can activate or inhibit CK1 depending on the substrate used

  • Assay conditions: Optimization of buffer composition, pH, and ion concentrations

The combination of using the BL21(DE3)pλPP expression system and careful attention to enzyme handling has been shown to consistently produce highly active CK1 preparations suitable for detailed biochemical and structural studies .

What are the critical factors affecting crystallization of recombinant CK1?

Successful crystallization of recombinant CK1 depends on several critical factors that researchers should consider:

• Protein homogeneity and purity:

  • Elimination of C-terminal degradation products: Expression in E. coli can produce heterogeneous preparations with 55-kDa full-length protein and degradation products of 50 and 42 kDa

  • Removal of flexible regions: The C-terminal autoinhibitory domain appears disordered in crystal structures

  • Monodispersity: Ensuring a uniform population of protein molecules

• Construct design decisions:

  • Truncation mutants: CK1δ lacking the C-terminal autoinhibitory region has been successfully crystallized

  • Full-length constructs: While crystallizable, the inhibitory domain may not be visible in electron density maps

  • Surface mutations: Modifying surface residues to promote crystal contacts

• Phosphorylation state:

  • Preventing autophosphorylation: Using expression systems with co-expressed phosphatases

  • Controlled phosphorylation: In some cases, specific phosphorylation states may promote crystallization

  • Verification of phosphorylation status: Ensuring batch-to-batch consistency

• Crystallization conditions:

  • Previous successful conditions: CK1δ crystals have been obtained in space group C2221

  • Resolution targets: Structures at 2.3-2.4 Å resolution have been achieved

  • Optimization strategies: Fine-tuning precipitant concentrations, pH, and additives

• Co-crystallization approaches:

  • ATP analogs: Inclusion of non-hydrolyzable ATP analogs can stabilize specific conformations

  • Substrate peptides: Co-crystallization with substrate peptides can provide insights into recognition mechanisms

  • Inhibitors: Co-crystallization with specific inhibitors like CKI-7

• Crystal handling and data collection:

  • Cryoprotection protocols: Proper cryoprotection to minimize radiation damage

  • Data collection strategies: Optimizing exposure times and crystal orientation

These factors should be systematically addressed to improve the chances of obtaining high-quality crystals suitable for structural studies of CK1.

What are the emerging applications of recombinant CK1 in research?

Recombinant CK1 continues to be a valuable tool in diverse research areas, with several emerging applications:

• Parasite biology and drug development:

  • CK1 family members play essential roles in the biology of major human protozoan parasites

  • The dual functions of CK1 in both parasite biology and host cell subversion make it an attractive target for anti-parasitic therapy

  • Targeting CK1 may increase the genetic barrier for the development of drug-resistant parasites

• Circadian rhythm research:

  • CK1's involvement in circadian rhythm regulation through phosphorylation of PER proteins makes recombinant CK1 valuable for studying these mechanisms

  • In vitro kinase assays with recombinant CK1 can help identify clock protein phosphorylation sites

• Wnt signaling studies:

  • CK1's role in phosphorylating β-catenin (CTNNB1) at Ser-45 positions it as a key regulator in Wnt signaling

  • Recombinant CK1 enables detailed biochemical characterization of this phosphorylation event

• Structural biology advances:

  • Ongoing improvements in expression and crystallization techniques may enable structural studies of full-length CK1 including regulatory domains

  • Cryo-electron microscopy approaches could provide insights into CK1 complexes with regulatory partners

• Development of specific inhibitors:

  • High-quality recombinant CK1 facilitates screening for isoform-specific inhibitors

  • Structure-based drug design approaches using crystallographic data

• Systems biology integration:

  • Incorporation of CK1 phosphorylation events into cellular signaling networks

  • Understanding the integration of CK1 activity with other kinases and phosphatases