α cdc37  (Cell Signaling Technology Inc)

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) HEK293 cells and A7r5 smooth muscle cells were lysed, and protein levels of BAG3 were assessed in relation to the housekeeping protein GAPDH using immunoblots. Quantitative analysis of BAG3 expression validated elevated expression in A7r5 cells. Statistical significance was evaluated through t tests, incorporating data from eight independent replicates. (B) Alignment of human, mouse, and rat BAG3 orthologs to visualize the conservation of the p-sites of interest. Functional p-site studies were carried out in these three organisms in preliminary studies and in rat and human in this work. (C) Summary of the phosphopeptides used in enzyme kinetic studies for the assessment of PP1c and PP5c-mediated dephosphorylation of BAG3 mimetics. (D) Monitoring of CDC37-pSer13 dephosphorylation in A7r5 lysate upon incubation with PP5, for the indicated incubation time to validate enzymatic activity. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of five independent experiments with adjusted P -values obtained from one-way ANOVA with Sidak correction (*** P = 0.0004), **** P < 0.0001). Mean is shown and error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, Functional Assay, De-Phosphorylation Assay, Incubation, Activity Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) HEK293 cells and A7r5 smooth muscle cells were lysed, and protein levels of BAG3 were assessed in relation to the housekeeping protein GAPDH using immunoblots. Quantitative analysis of BAG3 expression validated elevated expression in A7r5 cells. Statistical significance was evaluated through t tests, incorporating data from eight independent replicates. (B) Alignment of human, mouse, and rat BAG3 orthologs to visualize the conservation of the p-sites of interest. Functional p-site studies were carried out in these three organisms in preliminary studies and in rat and human in this work. (C) Summary of the phosphopeptides used in enzyme kinetic studies for the assessment of PP1c and PP5c-mediated dephosphorylation of BAG3 mimetics. (D) Monitoring of CDC37-pSer13 dephosphorylation in A7r5 lysate upon incubation with PP5, for the indicated incubation time to validate enzymatic activity. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of five independent experiments with adjusted P -values obtained from one-way ANOVA with Sidak correction (*** P = 0.0004), **** P < 0.0001). Mean is shown and error bars represent the SD. Source data are available for this figure.

Article Snippet: The membranes were blocked with Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% (wt/vol) non-fat milk at RT for 60 min. After three washes (5 min), the membranes were incubated overnight at 4°C with the following antibodies in TBS-T with 5% BSA: α-GAPDH (1:1,000, #2118), α-BAG3 (1:1,000, 23842S), and α-PP5 (1:1,000, 2289S), α-CDC37 (1:1,000, 3618S), α-CDC37-pS13 (1:1,000, 13,248), α-phosphoThr (1:500, 42H4, 9386), SQSTM1/p62 (1:1,000, 5114T), and α-14-3-3γ (1:500, 5522S) obtained from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, Functional Assay, De-Phosphorylation Assay, Incubation, Activity Assay

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: Specificity of the Polyclonal Anti-PhosphoSer13-Cdc37 Antibody (A) Coomasie-stained SDS-PAGE of purified H-C-K and C-K complexes. (B) Incubation of purified WT-Cdc37 expressed in E. coli with CK2 at 30°C shows a time-dependent specific signal from the α-pSer13-Cdc37 polyclonal antibody. No signal was observed with an S13A mutant. The blot was amido black-stained as a loading control. It has been shown elsewhere that Ser13 is the only site of phosphorylation by CK2 on Cdc37. (C) Free Cdc37, Cdc37 in complex with Cdk4 (C-K) and Cdc37 in complex with Hsp90 and Cdk4 (H-C-K) expressed in insect cells are all phosphorylated on Ser13.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: Staining, SDS Page, Purification, Incubation, Mutagenesis

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: Dephosphorylation of Free Cdc37 or Cdc37 within the C-K and H-C-K Complexes by λPP (A) The rate of dephosphorylation of free Cdc37, the C-K complex, and the H-C-K complex were compared. Samples with approximately equivalent amounts of Cdc37 were incubated in the absence (−) and presence (+) of λPP at room temperature for the specified times. Western blots were probed with α-pSer13-Cdc37. The blots were stripped and reprobed with α-Cdc37 as a loading control. (B) The C-K complex was incubated in the absence (−) and presence (+) of λPP and the effect of dephosphorylation on composition examined by analytical gel filtration with a Superose 6 10/300 GL column; 1 ml fractions were probed for each component of the complex by Western blot. Dephosphorylation was confirmed by probing with α-pSer13-Cdc37. Cdk4 alone elutes in fraction 19 (data not shown). (C) The H-C-K complex was incubated in the absence (−) or presence (+) of λPP. At specified time points, samples were withdrawn and the effect of dephosphorylation on complex stability assessed by pulldown of the complex via the His 6 -tagged Cdk4 on Talon resin. Samples were probed by Western blot for Cdc37 and Cdk4 and Coomassie stain for Hsp90. Dephosphorylation of the coprecipitated samples was confirmed by probing with α-pSer13-Cdc37. (D) The experiment was repeated in triplicate, and the amount of the components precipitated by the Talon resin (Bound) relative to the start of the incubation, was determined by densitometry of the Western blots for Cdk4 and Cdc37, or the Coomassie-stained PAGE gel for Hsp90. Error bars indicate SD of the measurements.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: De-Phosphorylation Assay, Incubation, Western Blot, Filtration, Staining

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: Dephosphorylation of Cdc37 by PP5 In Vitro (A) H-C-K complex or Sf9 -expressed Cdc37 was incubated with PP5 at room temperature and the extent of dephosphorylation at specified times assessed by probing Western blots with α-pSer13-Cdc37. This experiment was repeated in the presence of a 10-fold molar excess of PP5 activator—either a seven residue peptide (SRMEEVD) corresponding to the TPR binding motif of Hsp90 or full-length human Hsp90α. (B) The interaction of PP5 with Cdc37 is mediated by Hsp90. GST-tagged PP5 was incubated with Cdc37 or phosphorylated Cdc37 in the presence and absence of Hsp90 at 4°C. A small fraction of pSer13-Cdc37 is found in complex with GST-PP5, but the majority of Cdc37 remains phosphorylated; this interaction is significantly increased in the presence of Hsp90, and coincides with an increase in PP5 activity toward Cdc37 (Unbound, lanes 6 and 8). The affinity of GST-tagged PP5 for Hsp90-bound Cdc37 is low compared with that for Hsp90-bound pSer13-Cdc37 (10% Bound, lanes 7 & 8). CBB = Coomassie Brilliant Blue. (C) The interaction of PP5 with the H-C-K complex. GST-tagged PP5 was incubated with the H-C-K complex at 4°C, and could coprecipitate all components of the complex. The interaction was stabilized, and the activity of PP5 reduced, by inclusion of AMPPNP, but not ADP.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: De-Phosphorylation Assay, In Vitro, Incubation, Western Blot, Binding Assay, Activity Assay

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: Dephosphorylation of Cdc37p by Ppt1p in Yeast Extracts (A) Cdc37p is phosphorylated on both S14 and S17 in yeast cell extracts. Immunoprecipitated (IP) Cdc37-HA and its S14A and S17A mutants were immunoblotted with anti-Phospho-Serine antibody. (B) FLAG-tagged Ppt1p coimmunoprecipitates with the wild-type (WT), nonphosphorylatable (S14A/S17A), and phosphomimic (S14E/S17E) forms of HA-tagged Cdc37p. (C) HA-tagged wild-type Cdc37p immunoprecipitated from cells overexpressing FLAG-tagged Ppt1p is substantially dephosphorylated, as are the single S14A and S17A phosphorylation-site mutants. Overexpression of Ppt1 has no effect on the phosphorylation of HA-tagged Cdc37p in cells expressing the nonphosphorylatable S14A/S17A mutant. A FLAG-tagged Ppt1p K81E/R85E mutant, which abrogates Ppt1 interaction with Hsp90, no longer dephosphorylates HA-tagged Cdc37p.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Over Expression, Expressing, Mutagenesis

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: Effects of Cdc37 Mutation and Overexpression/Deletion of Ppt1 on v-Src Expression in Yeast (A) Growth of wild-type (CDC37) cells and strains with S14A, S17A, or the doubly mutated S14E/S17E Cdc37 containing the toxic vSrc gene controlled by the GAL promoter, on glucose (glu)- or galactose (gal)-containing medium. Both nonphosphorylatable and phosphomimetic mutants of Cdc37 compromise vSrc activation. (B) The same strains analyzed for total pY and v-Src levels. Appreciable levels of v-Src were only detectable in wild-type cells. (C) Both the loss ( ppt1Δ ) and overexpression ( PPT1 + ) of Ppt1p compromise v-Src expression in cells containing the toxic v-Src gene controlled by a GAL promoter. (D) The same strains analyzed for total pY and v-Src levels. As for Cdc37 phosphorylation mutants, appreciable levels of v-Src were only detectable in wild-type cells. (E) Overexpression of Ppt1p causes GA sensitivity in yeast. Cells expressing the wild-type (CDC37), nonphosphorylatable (S14A/S17A), or phosphomimic (S14E/S17E) Cdc37p were transformed with either empty vector or the PPT1 overexpression plasmid (MET25-PPT1). A 1:10 dilution series was grown (4 days, 30°C) on SD agar lacking methionine, without or with 30 μM GA. Overexpression of Ppt1 renders cells as sensitive as the nonphosphorylatable S14A, S17A mutant, of Cdc37.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: Mutagenesis, Over Expression, Expressing, Activation Assay, Transformation Assay, Plasmid Preparation

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: PP5 Dephosphorylates Cdc37 in Tumor Cells and Compromises C-Raf Function Cell lysate of HCT116 cells stably transfected with empty vector (v) or a PP5 expression plasmid (PP5). After 3 weeks, decreased levels of total C-Raf and activated ERK (phospho-ERK1/2) were observed. The effect was amplified further after an additional week of PP5 overexpression.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Amplification, Over Expression

Journal: Molecular Cell

Article Title: Hsp90-Dependent Activation of Protein Kinases Is Regulated by Chaperone-Targeted Dephosphorylation of Cdc37

doi: 10.1016/j.molcel.2008.07.021

Figure Lengend Snippet: Functional Cycle of Cdc37 Driven by Constitutive Phosphorylation and Targeted Dephosphorylation Schematic of coupling of CK2 phosphorylation and Hsp90-targeted dephosphorylation by PP5/Ppt1 to the role of Cdc37 in assembly and Hsp90-dependent activation of kinase clients. A C-K complex containing an inactive kinase binds an Hsp90 dimer with loss of one Cdc37 to give a stable H-C-K complex, as previously described ( Vaughan et al., 2006 ) (a). Cdc37 is phosphorylated in both these complexes (see above). PP5/Ppt1 can bind to Hsp90 in the H-C-K complex (b) and dephosphorylate Cdc37 prior to, or concomitant with, binding of ATP and other cochaperones, such as Aha1 (e.g., [c]), generating an activated kinase and dissociating the Hsp90-based complex. Free Cdc37 is rephosphorylated by the constitutive activity of CK2 (d) to regenerate the active pSer13 form of the cochaperone, poised for reengagement in a new round of Hsp90-dependent kinase activation.

Article Snippet: Other antibodies were: polyclonal α-Cdk4 (H303) (Santa Cruz Biotechnology); monoclonal α-Cdc37 (C1) (Affinity BioReagents); PP5 (C-20), Cdc37 (H-271), C-Raf (C-20) (Santa Cruz Biotechnology); P-ERK1/2 (no. 9101, Cell Signaling); total ERK2 (a gift of Chris Marshal, ICR); and GAPDH (Chemicon).

Techniques: Functional Assay, De-Phosphorylation Assay, Activation Assay, Binding Assay, Activity Assay