Fig 1: CDK6 inhibition increases tyrosine phosphorylation of proteins functioning in TCR signaling, and cyclin D3/CDK6 phosphorylates/activates PTP1B and TCPTP(A) GO enrichment analysis of TMT-MS data showed the top-ranked annotation groups among increased tyrosine-phosphorylated proteins in KOPTK1 cells upon treatment with palbociclib. 13 of 105 tyrosine-phosphorylated proteins relate to the TCR signaling pathway.(B) A list of PTPs, derived from TMT-MS data analysis, dephosphorylated at S/TP phosphorylation sites.(C) IB with anti-phospho-S/TP Abs of PTP1B or TCPTP immunoprecipitated from KOPTK1 cells treated with palbociclib (24 h).(D) IB with anti-phospho-S/TP Abs of immunoprecipitated PTP1B or TCPTP from interleukin-2 (IL-2)/CD3-activated splenic CD8+ T cells isolated from WT, CDK6 KO (center), or CDK4 KO mice (right) or from WT CD8+ T cells treated with vehicle or a CDK6 degrader (BSJ-03–123, 10 µM) for 24 h (left).(E) Phosphatase activities of PTP1B (left) and TCPTP (right) immunoprecipitated from Jurkat cells treated with a CDK4/6 inhibitor (palbociclib,1 µM) or CDK6 degrader (BSJ-03–123, 10 µM) for 24 h. Immunoblotting of PTP1B or TCPTP following immunoprecipitation (IP) shows protein loading of PTPs.(F) IB of recombinant PTP1B or TCPTP protein phosphorylated by cyclin D3/CDK6 following an in vitro kinase assay with anti-phospho-S/TP Abs. The recombinant Rb fragment was a positive control.(G) Phosphatase activities of PTP1B and TCPTP with or without cyclin D3/CDK6 phosphorylation.(H) IB with anti-phospho-S/TP Abs following kinase assays showed that PTP mutants were not or less phosphorylated by cyclin D3/CDK6 compared with the corresponding WT PTP.(I) Normalized phosphatase activities of PTP mutants following kinase assays with cyclin D3/CDK6.(E), (G), and (I) represent mean ± SEM; 1-way ANOVA (Dunnett’s test) (E and G) and two-tailed Student’s t test (I). n = 3 (E, G, and I). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 2: CDK6 inhibition increases CD3? tyrosine phosphorylation mediated by PTP1B and TCPTP(A–C) Immunoprecipitation using Abs specific to CD3e, CD3?, or CD3?, followed by immunoblotting using pY99 phosphotyrosine Abs to detect phosphorylation of CD3 subunits in Jurkat cells treated with palbociclib (24 h).(D) Comparative CD3? phosphorylation measured with flow cytometry by phospho-Tyr142 Abs in Jurkat cells with and without palbociclib treatment (24 h). n = 2.(E–G) In vitro phosphatase assay showed that CD3e, CD3?, and CD3? were the substrates of PTP1B and/or TCPTP.(H) In vitro phosphatase assay to detect the phosphatase activity of PTP1B or TCPTP immunoprecipitated from Jurkat cells treated with palbociclib (8 h). The phosphorylation level of PTP1B/TCPTP was detected using p-S/TP Abs.(I) IB using pY99 to detect phosphorylated CD3? immunoprecipitated from Jurkat cells treated with PTP1B-IN2 (1 µM, 24 h).(J) Phosphorylation levels of CD3? at Tyr142 in CD3/CD28/IL-2-activated splenic CD4+ or CD8+ T cells from CDK6 KO mice or WT littermates. n = 4.(K) IB of tyrosine phosphorylation of CD3? in CD8+ T cells from CDK6 KO mice or WT littermates or in WT CD8+ T cells treated with a CDK6 degrader for 48 h (L and M) Comparative levels of tyrosine phosphorylated CD3? (phospho-CD3? [Tyr142] Abs) in OVA peptide-activated CD8+ T cells isolated from OT-I mice (L) or primary human T cells derived from peripheral blood mononuclear cells (M) upon treatment with PTP1B-IN2 (1 µM), palbociclib (1 µM), or BSJ-03–123 (5 µM) for 24 h n = 4 (L). Con, n = 6; PTPIB-IN2, n = 7; palbociclib (Palbo), n = 7; CDK6 degrader, n = 6 (M).(N) Phosphorylation levels of CD3? at Tyr142 in human T cells upon genetic deletion of CDK6 (shCDK6) or cyclin D3 (shcyclinD3). n = 3.(D), (J), and (L)–(N) represent mean ± SEM; two-tailed Student’s t-test (D and J) and 1-way ANOVA (Dunnett’s test) (L–N); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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