lambda phosphatase  (New England Biolabs)

Journal: PLOS One

Article Title: Identification of CAND1 as a DNA-dependent protein kinase-regulated coactivator of androgen receptor and the ARv7 splice variant

doi: 10.1371/journal.pone.0349130

Figure Lengend Snippet: (A) After standard ARE pulldowns, the AR-DNA-bound complexes were washed and resuspended in 1x kinase buffer with/without ATP at 30 o C for listed times. Gel mobility shifts in AR were detected as a function of ATP addition. All pulldowns had 100 nM R1881 present. Proteins were analyzed by immunoblotting. Lanes 3 and 4 represent the standard AR-ARE pulldown. (B) To test if the above AR gel mobility shifts were reflective of phosphorylation events, the AR-CoR complexes were treated with ATP or AMP-PNP (non-hydrolyzable analogue) for 30 min at 30 o C. The DNA-PK inhibitor NU7441 or λ protein phosphatase (LPP) were pre-incubated for 5 min before adding ATP. LPP buffer was used for the LPP control reaction. (C) DNA-PK plays a role in phosphorylating AR. ATP treatment of the AR-CoR complexes using HeLa NE where DNA-PKcs was immunodepleted reveals a reduction of AR gel mobility shifts ( left panel) with the loss of DNA-PK. Proteins were assayed by immunoblotting and the level of unphosphorylated AR was quantified by Image J after normalization to the loading control HDAC1 ( right panel). P-value here = 0.00011. (D) DNA-bound AR or ARv7 was incubated with purified heterotrimeric DNA-PK (i.e., DNA-PKcs and Ku70/80) and then treated (-/+) ATP (-/+) NU7441 to demonstrate direct phosphorylation of AR/ARv7 by DNA-PK. Gel mobility shifts were assayed by immunoblotting. (E) ATP treatment (-/+) NU7441 pre-treatment demonstrates that DNA-PK enzymatic activity is needed for stabilizing CAND1 and ANP32A in the AR/ARv7 complexes. Auto-phosphorylation of DNA-PKcs at serine 2056 (pS2056) is a marker of DNA-PK activity and its inhibition by NU7441. Proteins were assayed by immunoblotting. ARv7 was detected here with the N-20 antibody. (F) HeLa NE used in 3xARE-E4 cell-free transcription assays was pre-treated (-/+) NU7441 to determine effects on AR and ARv7 transcriptional activity. E4 mRNA transcript levels were measured by RT-qPCR. In panels A-C and E, HDAC1 was used as a loading control and ARE pulldowns were conducted using HeLa NE as listed. All AR reactions had 100 nM R1881 present. Short exp., Medium exp., and Long exp. denote short, medium, and long exposures during x-ray film development. P-values: for ARv7, p = 0.00015; for AR, p = 0.000035. **** p < 0.0005, ***** p < 0.0001.

Article Snippet: DNA-PK inhibitor NU7441 was purchased from Tocris Bioscience. λ protein phosphatase (LPP) was purchased from New England Biolabs.

Techniques: Western Blot, Phospho-proteomics, Incubation, Control, Purification, Activity Assay, Marker, Inhibition, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: A tandem recruitment site in the pseudokinase scaffold PEAK3 is subject to phosphorylation-dependent regulation and cancer-associated mutations

doi: 10.1016/j.jbc.2026.111365

Figure Lengend Snippet: PEAK3 S69 phosphorylation changes dynamically upon insulin/EGF stimulation. A , schematic representation of PEAK3 structure, indicating pseudokinase domain and SH2, CrkII and 14-3-3 binding motifs. Multiple sequence alignment of vertebrate PEAK3 orthologues demonstrates conservation of the tandem CrkII/14-3-3 binding motif ( bottom panel ). The position of serine 69 (S69) is highlighted by an arrow . The pseudokinase domain is flanked by regulatory helices that form the split helical dimerization (SHED) domain (indicated by short black lines ). B , PEAK3 binding partners and downstream signaling pathways. Binding motifs for adaptor proteins Grb2 and CrkII, as well as 14-3-3, are embedded within the N-terminal region. SH2 domain-mediated recruitment of Grb2 to PEAK3 requires phosphorylation of tyrosine 24. Grb2 recruits the Arf GAP ASAP1 whilst CrkII is a known upstream regulator of Rac1. Binding of 14-3-3 to PEAK3 requires phosphorylation of serine 69 and functions as a negative molecular switch that attenuates PEAK3 signalling by inhibiting CrkII binding as well as PEAK3 tyrosine phosphorylation and Grb2 recruitment. C , specificity of the custom antibody against S69-phosphorylated PEAK3. Western blot analysis was performed on MCF-10A cells stably expressing HA-tagged PEAK3 or PEAK3 S69A, as indicated. Results are representative of at least three independent experiments. D , PEAK3 S69 phosphorylation is enhanced by EGF treatment. MCF-10A cells stably expressing HA-tagged PEAK3 were cultured in starvation medium overnight and then stimulated with EGF (5 min). Cell lysates were Western blotted as indicated. Cell lysates were also treated with lambda protein phosphatase to confirm that reactivity of the antibody is phosphorylation-dependent. Results are representative of three independent experiments. E , PEAK3 p-S69 dynamics upon EGF stimulation. MCF-10A/PEAK3 cells were stimulated with EGF for different times. Cell lysates were Western blotted as indicated. The densitometry analysis graph ( right ) indicates S69-phosphorylated PEAK3 normalized for HA-PEAK3 expression. Results are expressed relative to control (0 min) which is arbitrarily set at 1.0. Data represent the mean ± S.E.M. of n = 3 independent experiments with 'au' indicating arbitrary units. Statistical significance was assessed by ratio paired t test, two tailed. ∗∗ p < 0.01, ns , not significant. F , PEAK3 p-S69 dynamics upon insulin stimulation. Experiments and data analysis were performed as described in E , except that cells were stimulated with insulin. Data represent the mean ± S.E.M. of three independent experiments. ∗ p < 0.05, by ratio paired t test, two tailed. G , impact of dimerization on PEAK3 S69 phosphorylation. HA-tagged PEAK3 or dimerization-interface mutants (L146E, A436E, C453E) were overexpressed in PEAK1/2 double knockout (DKO) MCF-10A cells and S69 phosphorylation was analyzed by Western blotting. Results are representative of two independent experiments.

Article Snippet: The following reagents/inhibitors were used in this study: lambda protein phosphatase (New England Biolabs, P0753S), animal-free recombinant human EGF (PeproTech, AF-100–15), insulin from bovine pancreas (Merck, I-1882), CaMKII inhibitor RA306 (custom synthesized by Reagency, RGNCY-0117, 1 μM final), PKA Inhibitor 14 to 22 Amide, Cell-Permeable, Myristoylated (Calbiochem, 476,485, 10 μM final), DMSO (Sigma, d8418, 0.1% final), and MedChemExpress-sourced Akt inhibitor MK-2206 dihydrochloride (HY-10358, 100 nM final) and trametinib (HY-10999, 10 nM final).

Techniques: Phospho-proteomics, Binding Assay, Sequencing, Protein-Protein interactions, Western Blot, Stable Transfection, Expressing, Cell Culture, Control, Two Tailed Test, Double Knockout