Phospho-CDC37 (Ser13) Antibody

What is CDC37 and what is the significance of its phosphorylation at Ser13?

CDC37 functions as a co-chaperone protein that collaborates with Heat Shock Protein 90 (HSP90) to facilitate the folding, maturation, and stabilization of protein kinases. CDC37 is phosphorylated by Casein Kinase II (CKII or CK2) at its Ser13 residue; this phosphorylation is required for its interaction with HSP90 and target protein stabilization function . The modification is critical for CDC37's ability to coordinate HSP90 nucleotide-mediated conformational switching and kinase binding, as demonstrated through mutation studies where Ser13 mutations to either Ala or Glu compromised the recruitment of CDC37 to HSP90-kinase complexes . Multiple studies have confirmed that complexes of HSP90 with client signaling kinases (including Cdk4, MOK, v-Src, and Raf1) contain the CK2-phosphorylated form of CDC37 in vivo . This phosphorylation represents a critical regulatory mechanism in kinase chaperoning pathways and potential therapeutic targets in diseases where kinase dysregulation occurs.

How does phosphorylation at Ser13 affect the structure and function of CDC37?

Phosphorylation at Ser13 induces significant structural and functional changes in CDC37 that are essential for its chaperoning activity. Structurally, phosphorylation induces a large shift toward a more compact conformation, as demonstrated by ANS fluorescence experiments . This modification modestly increases secondary structure while requiring interactions between the N-terminal domain and the remainder of CDC37 for the structural transition to occur . Interestingly, the stabilizing property of phosphorylation can be recreated in trans by a phospho-Ser13 peptide derived from the N-terminal tail .

Functionally, this phosphorylation is essential for CDC37's proper interaction with HSP90 and its target protein stabilization function . The modification is critical for the recruitment of CDC37 to HSP90-kinase complexes, while having only modest effects on CDC37's basal (client-free) binding to HSP90 . CDC37 with Ser13Glu mutations (phosphomimetic) showed altered interactions with HSP90-kinase complexes, suggesting compromised modulation of the HSP90 ATP-driven reaction cycle . These structural and functional changes explain how this post-translational modification regulates CDC37's ability to mediate protein-protein interactions in kinase stabilization pathways.

What techniques can be used to detect CDC37 phosphorylation at Ser13?

Multiple validated techniques are available for detecting and analyzing CDC37 phosphorylation at Ser13:

For Western blotting, phospho-specific antibodies selectively recognize only the phosphorylated form of CDC37 at Ser13 . Immunoprecipitation can be performed with these antibodies to study protein complexes containing phosphorylated CDC37 . Phospho-affinity gel electrophoresis takes advantage of the fact that CK2-dependent phosphorylation of CDC37 on Ser13 causes a specific gel mobility shift, allowing distinction between phosphorylated and non-phosphorylated forms . Cell-Based ELISAs enable qualitative determination of phospho-CDC37 levels in intact cells under different experimental conditions . Finally, immunofluorescence microscopy has revealed that phosphorylated CDC37 accumulates in specific cellular locations, such as epidermal growth factor-induced membrane ruffles .

What are the recommended controls when using Phospho-CDC37 (Ser13) antibody?

Implementing appropriate controls is essential for interpreting results when using phospho-specific antibodies:

Positive controls:

• Cell lysates from lines known to express phosphorylated CDC37 (e.g., K562 cells)

• Recombinant CDC37 phosphorylated in vitro with CK2

• Samples treated with agents that enhance CK2 activity

Negative controls:

• Phosphatase-treated samples to remove phosphorylation

• CDC37 Ser13Ala mutant expressed in cells

• Samples treated with CK2 inhibitors

Loading and normalization controls:

• Total CDC37 antibody should be used on parallel blots or after stripping and reprobing

• GAPDH as an internal positive control for normalizing target values

• Crystal Violet whole-cell staining for cell density determination in cell-based assays

For immunoprecipitation experiments, additional controls should include an IgG control antibody from the same species as the phospho-specific antibody and control immunoprecipitation with non-specific antibody . Input sample (pre-IP) should be included to confirm the presence of the protein in starting material . Multiple normalization methods are particularly important for Cell-Based ELISAs, where antibodies against non-phosphorylated counterparts can be used for normalization of phosphorylated targets .

How can I verify the specificity of a Phospho-CDC37 (Ser13) antibody?

Verifying antibody specificity is crucial for reliable research results. Several approaches can be used to confirm the specificity of Phospho-CDC37 (Ser13) antibodies:

Phosphatase treatment control:

• Treat a portion of your sample with lambda phosphatase or calf intestinal alkaline phosphatase

• A genuine phospho-specific antibody will show significantly reduced or abolished signal in phosphatase-treated samples

In vitro phosphorylation assays:

• Incubate recombinant CDC37 with CK2 in the presence of Mg²⁺ and ATP

• The antibody should recognize CDC37 only after this treatment

Mutational analysis:

• Express CDC37 with Ser13 mutated to a non-phosphorylatable residue (e.g., Ser13Ala)

• The antibody should not recognize these mutant versions

Cross-reactivity testing:

• Test the antibody against other CK2 substrates such as HSP90 and FK506-binding protein 52

• A specific antibody should not cross-react with these other phosphoproteins

Immunodepletion approach:

• Perform sequential immunodepletions using the phospho-specific antibody

• Western blot analysis with a total CDC37 antibody can reveal depletion efficiency

One study demonstrated specificity by showing their phospho-specific antibody recognized recombinant purified CDC37 only when incubated with CK2 in the presence of Mg²⁺ and ATP, and that replacement of Ser13 with nonphosphorylatable amino acids abolished binding .

What are the best methods for immunodepletion of phosphorylated CDC37?

Immunodepletion is valuable for estimating phosphorylation efficiency in vitro and in cell culture. Based on published protocols, here is a detailed method for CDC37 phospho-Ser13 immunodepletion:

Materials needed:

• CDC37 phospho-Ser13 specific antibody (e.g., Epitomics #3600-1)

• Protein A/G agarose beads (e.g., Santa Cruz sc-2003)

• PBS buffer

• Total CDC37 antibody for detection (e.g., Santa Cruz sc-13129)

Sequential immunodepletion procedure:

• Dilute CDC37-containing solutions (either in vitro phosphorylation reaction or cell lysate) into 500 μL of PBS

• Add 4 μL of CDC37 phospho-S13 antibody

• Add 15 μL of protein A/G agarose beads

• Incubate on a rotator for 30 minutes at room temperature

• Centrifuge to pellet the beads and collect the supernatant

• Repeat steps 2-5 for a second and third round of immunodepletion

• Analyze the remaining soluble CDC37 by Western blot using a total CDC37 antibody

This sequential approach ensures more complete depletion of phosphorylated CDC37, allowing for more accurate quantification of phosphorylation ratios. The multiple rounds of depletion are particularly important when studying samples with high levels of phosphorylated CDC37. For studying cellular interactions of recombinant CDC37 with client proteins, modified protocols incorporate additional purification steps like glutathione agarose resin and gel filtration spin columns for rebuffering before analysis .

How does Phospho-CDC37 (Ser13) interact with HSP90 and client kinases?

The interaction between phosphorylated CDC37, HSP90, and client kinases has been characterized through several experimental approaches:

Co-immunoprecipitation studies reveal that complexes of HSP90 with client signaling kinases (Cdk4, MOK, v-Src, and Raf1) contain the CK2-phosphorylated form of CDC37 in vivo . Phospho-affinity gel electrophoresis demonstrates that CDC37 in complexes between HSP90 and its client signaling protein kinases is predominantly in the phosphorylated form . Immunofluorescence microscopy shows that HSP90 and phosphorylated CDC37 co-localize in specific cellular compartments, such as epidermal growth factor-induced membrane ruffles, suggesting functional interactions in response to signaling events .

Mutational analysis reveals that mutation of Ser13 to either Ala or Glu compromises the recruitment of CDC37 to HSP90-kinase complexes while having only modest effects on CDC37's basal (client-free) binding to HSP90 . CDC37 with Ser13Glu mutations showed altered interactions with HSP90-kinase complexes, suggesting compromised modulation of the HSP90 ATP-driven reaction cycle . These findings indicate that phosphorylation of CDC37 at Ser13 is critical for coordinating HSP90 nucleotide-mediated conformational switching and kinase binding, highlighting the importance of this post-translational modification in kinase chaperoning.

How do mutations near the Ser13 site affect CDC37 phosphorylation and function?

Mutations in the vicinity of Ser13 have significant effects on CDC37 phosphorylation and function, providing insights into the structural requirements for proper CDC37 activity:

Several critical residues near Ser13 (His9, Glu11, and Asp14) form part of the highly conserved N-terminal region of CDC37 that is required for client kinase binding . Mutations in these residues disrupt the feedback mechanism between CK2 and CDC37, diminishing or preventing phosphorylation of CDC37 by CK2 . These mutations prevent recruitment of CK2 to CDC37 despite not overlapping with the CK2 consensus sequence (SXXE/D) . As a result, recruitment of client kinases (ULK1, Raf-1, Cdk4) is affected because their association depends on CDC37 phosphorylation .

Direct Ser13 mutations to either Ala or Glu compromise the recruitment of CDC37 to HSP90-kinase complexes while having only modest effects on CDC37's basal (client-free) binding to HSP90 . CDC37 with Ser13Glu mutation (phosphomimetic) showed altered interactions with HSP90-kinase complexes consistent with compromised CDC37 modulation of the HSP90 ATP-driven reaction cycle . These findings highlight the essential nature of the polypeptide sequence surrounding CDC37-Ser13 in client kinase activation , suggesting that the structural integrity of this region is critical for proper CDC37 function beyond just the phosphorylation event itself .

What is the relationship between PP5 and CDC37 phosphorylation?

Protein Phosphatase 5 (PP5) plays a critical role in regulating CDC37 phosphorylation, representing an important regulatory mechanism in kinase chaperoning:

PP5 can dephosphorylate CDC37 at Ser13, as revealed by the crystal structure of PP5 with CDC37 trapped in the active site . This structure shows how PP5 associates with CDC37 as a substrate . PP5's catalytic activity is crucial for this dephosphorylation, as mutations in the catalytic cleft (N308D, M309C, Y313F, and W386F) strongly diminish dephosphorylation of phospho-CDC37-Ser13 .

This relationship can be experimentally manipulated through several approaches:

• Genetic manipulation using siRNA knockdown of PP5 affects CDC37 phosphorylation levels , while overexpression of PP5 enhances CDC37 dephosphorylation

• Structural studies using a chimeric protein comprising the catalytic domain of PP5 (residues 175-499) and a peptide from CDC37 (residues 5-20, with S13E mutation) joined by a flexible linker

• Specific residue manipulation through mutations in PP5's catalytic cleft to selectively inhibit PP5-mediated CDC37 dephosphorylation

• Direct interaction studies via reciprocal co-immunoprecipitation experiments showing direct interaction between PP5 and CDC37

Structural changes are required for PP5-mediated dephosphorylation of CDC37 in the context of client-loaded HSP90 chaperone complex , and this may be the trigger for client kinase release from the HSP90-chaperone complex .

How can phospho-affinity gel electrophoresis be optimized for CDC37 studies?

Phospho-affinity gel electrophoresis is a powerful technique for studying CDC37 phosphorylation states, offering several advantages:

• Demonstrates that CK2-dependent phosphorylation of CDC37 on Ser13 causes a specific gel mobility shift

• Allows visualization of phosphorylated vs. non-phosphorylated forms in the same sample

• Confirms that CDC37 in HSP90-client kinase complexes is predominantly phosphorylated

Optimization strategies:

• Sample preparation:

  • For in vitro phosphorylation: Incubate recombinant CDC37 with CK2 in buffers containing Mg²⁺ and ATP

  • For cellular samples: Quick lysis with phosphatase inhibitors preserves phosphorylation states

  • Include phosphatase-treated controls to confirm band shifts are phosphorylation-dependent

• Gel composition:

  • Incorporate Phos-tag™ acrylamide at optimized concentrations (25-100 μM)

  • Include Mn²⁺ or Zn²⁺ ions; test both to determine optimal separation

  • Adjust polyacrylamide percentage based on CDC37's molecular weight (~50 kDa)

• Running conditions:

  • Use lower voltage (100V) with longer run times for better separation

  • Test different buffer compositions and pH values for optimal resolution

  • Maintain temperature control during runs for reproducibility

• Detection methods:

  • Prior to transfer, soak gels in EDTA-containing buffer to remove metal ions

  • Use both phospho-specific and total CDC37 antibodies on parallel blots

  • Validate shifted bands with phospho-specific antibodies

This technique has proved valuable for demonstrating the physiological importance of CK2-dependent CDC37 phosphorylation and can be adapted to study various aspects of CDC37 regulation in different experimental contexts .

How can I study the dynamics of CDC37 Ser13 phosphorylation in living cells?

Studying the dynamics of CDC37 phosphorylation in living cells requires specialized approaches:

Real-time monitoring approaches:

• Design FRET-based biosensors with fluorescent protein pairs flanking the CDC37 Ser13 region that undergo conformational changes upon phosphorylation

• Develop cell-permeable fluorescent probes that specifically bind to phosphorylated CDC37

Inducible expression systems:

• Create cell lines with inducible expression of CK2 or PP5 to control the phosphorylation/dephosphorylation cycle

• Employ rapid-acting chemical inducers for temporal control of enzyme activity

Pulse-chase experiments:

• Use metabolic labeling with ³²P followed by immunoprecipitation of CDC37 to track phosphorylation turnover rates

• Chase with phosphatase activators or CK2 inhibitors to measure dephosphorylation kinetics

Phosphorylation regulator manipulation:

• siRNA knockdown of PP5 affects CDC37 phosphorylation levels

• Overexpression of PP5 enhances CDC37 dephosphorylation

• CK2 inhibitors can block new phosphorylation events

Subcellular localization:

• Immunofluorescence studies show HSP90 and phosphorylated CDC37 accumulate in epidermal growth factor-induced membrane ruffles

• Live-cell imaging with fluorescently tagged proteins can track phosphorylated CDC37 movement

By combining these approaches, researchers can gain insights into the spatiotemporal regulation of CDC37 phosphorylation and its role in kinase chaperoning pathways.

What experimental approaches can investigate CDC37 Ser13 phosphorylation in kinase chaperoning?

To investigate how CDC37 Ser13 phosphorylation affects specific kinase chaperoning pathways, several sophisticated approaches can be employed:

Client kinase-specific interaction studies:

• Co-immunoprecipitation experiments have shown that complexes of HSP90 with client kinases like Cdk4, MOK, v-Src, and Raf1 contain phosphorylated CDC37

• Develop similar approaches for your kinase of interest using antibodies against both the kinase and phospho-CDC37

Mutational approaches:

• Express CDC37 Ser13Ala or Ser13Glu mutants and assess effects on specific kinase stability and activity

• Examine downstream signaling pathway activation after expression of these mutants

Kinase activity assays:

• Measure activity of specific client kinases with wild-type vs. phospho-deficient CDC37

• Correlate CDC37 phosphorylation state with kinase activity levels

Client kinase folding and maturation:

• Use pulse-chase experiments to track newly synthesized kinase folding rates

• Employ protease sensitivity assays to assess structural integrity of client kinases

Pathway-specific reporters:

• Utilize luciferase or fluorescent reporters downstream of specific kinase pathways

• Measure functional outcomes of CDC37 phosphorylation manipulation

Studies have shown that the N-terminal region of CDC37 including Ser13 is essential for client kinase binding , and mutations in this region (His9, Glu11, and Asp14) disrupt CK2 recruitment to CDC37, preventing phosphorylation and affecting recruitment of client kinases like ULK1, Raf-1, and Cdk4 .

What are key considerations when investigating the CK2-CDC37-HSP90 regulatory axis?

Investigating the CK2-CDC37-HSP90 regulatory axis requires careful experimental design to address the complex interplay between these components:

1. Phosphorylation state analysis:

• CK2 constitutively phosphorylates CDC37 at Ser13 in cells

• This phosphorylation creates a more stable and compact conformation of CDC37

• Use phospho-specific antibodies or phospho-affinity gel electrophoresis to monitor phosphorylation status

2. Functional feedback mechanisms:

• A positive feedback loop exists between CK2 and CDC37 that regulates CK2 activation and CDC37 phosphorylation

• His9, Glu11, and Asp14 in CDC37 are involved in this feedback mechanism

• Mutations in these residues disrupt feedback by preventing CK2 recruitment to CDC37

3. Structural considerations:

• Phospho-Ser13 CDC37 has a more stable and compact conformation than non-phosphorylated CDC37

• This conformation is accessible to dephosphorylation by phosphatases

• For client kinases like B-Raf, this conformation enhances complex stability but isn't required for client recognition

4. Domain-specific interactions:

• The primary interaction of CDC37 with client kinases occurs through a C-terminal domain of CDC37, remote from the Ser13 phosphorylation site

• Both N-terminal and C-terminal interactions should be examined in experimental design

5. Dephosphorylation regulation:

• PP5 dephosphorylates CDC37 at Ser13

• Structural changes are required for PP5-mediated dephosphorylation in client-loaded HSP90 complexes

• This may trigger client kinase release from the HSP90-chaperone complex

These considerations will help design robust experiments to investigate the complex regulatory mechanisms governing the CK2-CDC37-HSP90 axis in kinase chaperoning pathways.