CDC37 Human, His

What is CDC37 and what is its primary function in human cells?

CDC37 (Cell Division Cycle 37 homolog) is a molecular chaperone with a specialized role in cell signal transduction. It forms complexes with Hsp90 and various protein kinases including CDK4, CDK6, SRC, RAF-1, MOK, and eIF2 alpha kinases. CDC37's primary function is directing Hsp90 to its target kinases, where it plays a crucial role in their stability, folding, and activation .

The protein acts as a co-chaperone that recruits immature client kinases to Hsp90 for proper folding. The catalytic domains of these kinases are stabilized by CDC37, and their proper folding and functioning is dependent on Hsp90 . In experimental settings, CDC37 has been shown to facilitate complex assembly between CDK4 and cyclin D1, demonstrating its role in promoting protein-protein interactions essential for cell cycle regulation .

What is the significance of the His-tag in CDC37 Human, His protein for research applications?

The His-tag in CDC37 Human, His refers to a 10 amino acid N-terminal histidine tag (MKHHHHHHAS) that is fused to the 388 amino acid CDC37 protein, resulting in a 45.71 kDa recombinant protein . This tag serves several critical methodological functions in research:

• Purification: The His-tag allows for simple and efficient purification using metal affinity chromatography

• Detection: Facilitates convenient western blot detection using anti-His antibodies

• Immobilization: Enables protein immobilization for interaction studies

• Tracking: Provides a consistent means to track the protein in various experimental systems

When designing experiments, researchers should consider that while the His-tag generally has minimal impact on protein function, it may occasionally affect protein folding or interactions in specific experimental contexts. Control experiments comparing tagged and untagged versions may be necessary for definitive studies .

How does the domain structure of CDC37 relate to its function?

CDC37's domain structure directly correlates with its functional specialization:

The N-terminal domain of CDC37 is sufficient for binding to client protein kinases like v-Src, as demonstrated through truncation experiments with CDC37 1-173 . The middle domain contains the Hsp90 binding site, with Leu-205 identified as a key residue enabling complex formation with Hsp90 . This modular domain architecture allows CDC37 to simultaneously bind both client kinases and Hsp90, functioning as a molecular bridge in chaperoning complexes .

In yeast studies, the protein kinase-binding domain of CDC37 was sufficient for cell viability and permitted efficient signaling through MAP kinase pathways, indicating the evolutionary conservation of this critical functional domain .

What are the optimal conditions for reconstituting lyophilized CDC37 Human, His protein?

To properly reconstitute lyophilized CDC37 Human, His protein for experimental use, follow this methodological approach:

• Add deionized water to the lyophilized product to prepare a working stock solution of 0.5 mg/ml

• Allow the lyophilized pellet to dissolve completely at room temperature with gentle agitation

• For cell culture applications, filter the reconstituted protein through an appropriate sterile filter (0.22μm) as the product is not provided sterile

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• For short-term storage (up to two weeks), store at 4°C

• For long-term storage, keep aliquots at -20°C

The reconstituted protein maintains stability at 4°C for approximately two weeks without significant degradation . For experimental reproducibility, it's advisable to use freshly prepared aliquots whenever possible and avoid multiple freeze-thaw cycles that may compromise protein structure and function.

How can researchers effectively study CDC37-client protein interactions in experimental systems?

Studying CDC37-client protein interactions requires specialized methodological approaches to capture these often transient and complex relationships:

• Yeast Two-Hybrid System: This approach successfully identified CDC37 interaction with CDK4 and CDK6, but not with CDC2, CDK2, CDK3, CDK5, or various cyclins, demonstrating its specificity for particular kinases . The methodology involves:

  • Fusion of CDC37 to DNA-binding domain

  • Fusion of potential client proteins to activation domain

  • Co-transformation into yeast cells

  • Selection on appropriate media and β-galactosidase assays

• Co-immunoprecipitation with Recombinant Proteins: Using purified full-length or truncated CDC37 proteins with in vitro synthesized labeled potential clients. This method revealed that:

  • Full-length CDC37 binds both Hsp90 and v-Src

  • Truncated CDC37 (1-173) lacking the Hsp90-binding site retained v-Src binding

• Competitive Binding Assays: These revealed that CDC37 competes with p16 for binding to CDK4, suggesting a mechanism for p16's inhibitory function through disruption of CDK4/cyclin D1 complex formation .

For optimal results, researchers should employ multiple complementary approaches and include appropriate controls for validation of specific interactions.

What experimental design considerations are important when using CDC37 Human, His in protein folding studies?

When designing protein folding studies with CDC37 Human, His, researchers should consider several critical methodological factors:

• Buffer Composition: CDC37 Human, His is typically supplied in 20mM TRIS, 50mM NaCl, pH 7.5 . This buffer provides stability while maintaining compatibility with most folding assays. Consider buffer requirements of client proteins when designing experiments.

• Client Kinase Selection: CDC37 exhibits specificity for certain kinases (CDK4, CDK6, v-Src) but not others (CDC2, CDK2, CDK3, CDK5) . Select appropriate client kinases based on documented interactions.

• Chaperone Complex Reconstitution: Since CDC37 functions in concert with Hsp90, experimental systems should include:

  • Purified Hsp90

  • ATP and appropriate cofactors

  • Client kinase substrate

  • Additional cochaperones as needed for complete systems

• Temperature and Time Course: CDC37-mediated folding is temperature-dependent; conduct experiments at physiologically relevant temperatures (30-37°C) with appropriate time points to capture the kinetics of folding.

• Controls: Include critical controls such as:

  • CDC37 mutants lacking kinase-binding domain

  • ATP-depleted conditions

  • Inhibitors of Hsp90 (geldanamycin) to distinguish CDC37-dependent vs. Hsp90-dependent effects

By carefully considering these factors, researchers can develop robust experimental systems for studying CDC37's role in protein folding.

How does CDC37 contribute to cancer development and progression?

CDC37 plays multiple mechanistic roles in cancer development and progression, as evidenced by its upregulation in human prostate cancer:

• Proliferation Enhancement: Overexpression of CDC37 drives proliferation in normal prostate epithelial cells. In human prostate tissue specimens, increased CDC37 immunoreactivity was found in all prostate cancer specimens analyzed when compared with normal tissues .

• Apoptosis Resistance: Loss of CDC37 function leads to growth arrest and apoptosis, suggesting that its overexpression in cancer contributes to cell survival mechanisms .

• Signaling Pathway Dysregulation: CDC37 overexpression leads to:

  • Enhanced Raf-1 activity

  • Greater CDK4 levels

  • Reduced expression of the cyclin-dependent kinase inhibitor p16/CDKN2

• Early Cancer Development: CDC37 overexpression was found in the luminal cells of prostate cancer precursor lesion (PIN), indicating its activation is an early event in cancer development .

• Client Kinase Stabilization: CDC37 stabilizes numerous oncogenic kinases, maintaining their function in cancer cells and supporting their continued signaling .

These findings suggest CDC37 as a potential therapeutic target in cancer treatment strategies, particularly through disruption of its interactions with client kinases or Hsp90 .

What role does CDC37 play in wound healing processes?

CDC37 has been shown to play critical roles in wound healing processes, particularly in Drosophila larval epidermis where it mediates both cell growth and migration:

• JNK Pathway Stabilization: CDC37 maintains the stability of JNK pathway kinases (JNK, Hep, Mkk4, and Tak1), which are essential for wound closure. In larvae lacking CDC37 in the epidermis:

  • The JNK pathway was not typically activated following injury

  • Protein levels of JNK pathway components were reduced

  • Wounds failed to close properly

• Cell Morphology Regulation: Cells in CDC37 knockdown larvae failed to:

  • Change cell shape

  • Properly polarize

  • Undergo wound-induced cell growth

• Integrin Expression: CDC37 affects the expression of integrin beta subunit, which is crucial for proper cell migration during wound healing. CDC37 knockdown resulted in:

  • Reduced protein levels of integrin beta

  • Diminished wound-induced protein expression of integrin beta

This research demonstrates CDC37's importance beyond cancer, highlighting its fundamental role in basic cellular processes required for tissue repair through its chaperone function for key signaling kinases .

How might researchers exploit the structural features of CDC37-Hsp90 interaction for targeted drug development?

The structural elucidation of the CDC37-Hsp90 interaction provides valuable insights for targeted drug development:

• Key Interaction Sites: The x-ray crystal structure of the 16-kDa middle domain of human CDC37 at 1.88 Å resolution and its complex with the 23-kDa N-terminal domain of human Hsp90 reveals specific interaction surfaces . Drug development could focus on:

  • Disrupting the CDC37-Hsp90 interface

  • Targeting the Leu-205 residue in CDC37, identified as critical for complex formation

  • Designing molecules that induce conformational changes in the middle domain of CDC37

• Monomer-Specific Targeting: NMR studies demonstrate that the middle domain of CDC37 exists as a monomer, suggesting monomer-specific binding pockets for small molecule inhibitors .

• Selective Disruption Strategy: Unlike broad Hsp90 inhibitors that affect numerous clients, CDC37-targeted drugs could selectively affect kinase clients while sparing other Hsp90 dependencies . This approach could potentially reduce toxicity while maintaining efficacy in kinase-dependent cancers.

• Combinatorial Approaches: Targeting both CDC37-client interaction and CDC37-Hsp90 interaction simultaneously could provide synergistic effects. Research models suggest CDC37 can function independently of Hsp90 when overexpressed, indicating that dual targeting may be necessary for complete inhibition .

These structural insights provide rational design principles for developing small molecule inhibitors against cancer through disruption of the CDC37-Hsp90-kinase axis .

What are the mechanistic differences in CDC37 function between normal and cancer cells?

The mechanistic differences in CDC37 function between normal and cancer cells represent a critical area of investigation:

• Expression Level Regulation:

  • Normal cells: CDC37 is expressed at very low levels and is not inducible under conditions that up-regulate other chaperones and heat shock proteins

  • Cancer cells: CDC37 is consistently upregulated, particularly in prostate cancer where increased immunoreactivity is found in all specimens analyzed compared to normal tissues

• Client Kinase Processing Pathway:

  • Normal cells: CDC37 functions in a coordinated "triage" involving other chaperones such as Hsp70 before accessing client kinases

  • Cancer cells: Elevated CDC37 may bypass this triage system, potentially allowing direct chaperoning of kinases independent of the normal regulatory controls

• Hsp90-Dependent vs. Independent Functions:

  • Normal cells: CDC37 primarily functions in conjunction with Hsp90

  • Cancer cells: Overexpression of CDC37 may enable Hsp90-independent functions, as demonstrated in yeast models where CDC37 truncation mutants deleted for the Hsp90-binding site still stabilized v-Src and led to some folding

• Downstream Pathway Alterations:

  • In prostate epithelial cells, CDC37 overexpression leads to enhanced Raf-1 activity, greater CDK4 levels, and reduced p16/CDKN2 expression, creating a pro-proliferative state characteristic of cancer

Understanding these mechanistic differences provides insights into how CDC37 contributes to cancer development and potential vulnerabilities that could be targeted therapeutically.

How does CDC37 function in an Hsp90-independent manner, and what are the implications for cellular regulation?

Evidence suggests CDC37 can function independently of Hsp90 under certain conditions, which has significant implications for understanding cellular regulation:

• Experimental Evidence for Hsp90-Independent Function:

  • CDC37 truncation mutants lacking the Hsp90-binding site still stabilized v-Src and led to some folding in both sti1Δ and hsc82Δ yeast strains

  • The protein kinase-binding domain of CDC37 alone was sufficient for yeast cell viability and permitted efficient signaling through the MAP kinase pathway

• Proposed Mechanisms of Independent Function:

  • Direct stabilization of kinase structure through interaction with the catalytic domain

  • Prevention of aggregation through chaperone activity independent of Hsp90

  • Facilitation of complex assembly between kinases and their binding partners (e.g., CDK4 and cyclin D1) without Hsp90 involvement

• Regulatory Implications:

  • CDC37's normally low expression levels may restrict its independent function, creating a form of regulation

  • This may establish a regulatory checkpoint requiring clients to pass through a triage involving other chaperones like Hsp70 before accessing CDC37

  • Overexpression of CDC37 (as in cancer) may bypass this regulatory mechanism

• Competition with Inhibitory Proteins:

  • CDC37 competes with p16 for binding to CDK4, suggesting an additional regulatory mechanism

  • This competition may affect the formation of CDK4/cyclin D1 complexes independent of Hsp90 function

This Hsp90-independent function of CDC37 represents an important paradigm shift in understanding chaperoning networks and may explain why certain CDC37-dependent processes are resistant to Hsp90 inhibitors, with significant implications for therapeutic strategies.

What are common technical challenges when working with CDC37 Human, His and how can they be addressed?

Researchers working with CDC37 Human, His may encounter several technical challenges that require specific methodological solutions:

• Protein Stability Issues:

  • Challenge: CDC37 may show degradation during storage or experimental manipulation

  • Solution: Store lyophilized CDC37 at -20°C and aliquot after reconstitution to avoid repeated freezing/thawing cycles. Reconstituted CDC37 can be stored at a maximum of 4°C for up to two weeks

• Solubility Problems:

  • Challenge: Incomplete dissolution of lyophilized protein

  • Solution: Add deionized water to prepare a working stock solution of 0.5 mg/ml and ensure the lyophilized pellet dissolves completely before use. Gentle agitation without vigorous vortexing can help maintain protein integrity

• Complex Formation Difficulties:

  • Challenge: Inconsistent formation of CDC37-client complexes in experimental systems

  • Solution: Optimize buffer conditions (20mM TRIS, 50mM NaCl, pH 7.5 is recommended), and ensure proper stoichiometry between CDC37 and client proteins

• His-Tag Interference:

  • Challenge: The His-tag may occasionally interfere with specific interactions

  • Solution: Consider using tag-cleavable constructs with protease sites for critical experiments where tag interference is suspected

• Client Specificity Issues:

  • Challenge: Variability in CDC37 binding to different kinases

  • Solution: Remember CDC37 shows specificity for certain kinases (CDK4, CDK6) but not others (CDC2, CDK2, CDK3, CDK5), so select appropriate experimental systems

Addressing these technical considerations will improve experimental reproducibility and data quality when working with CDC37 Human, His.

How can researchers reconcile contradictory data on CDC37 function from different experimental systems?

Reconciling contradictory data on CDC37 function requires careful consideration of several factors that may influence experimental outcomes:

By systematically analyzing these factors, researchers can better integrate seemingly contradictory findings into a more comprehensive understanding of CDC37 function across different biological contexts.