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A PTPB1-Cdk3 signaling axis promotes cell cycle progression of human glioblastoma cells through an Rb-E2F dependent pathway

ABSTRACT: Protein tyrosine phosphatase 1B (PTP1B) plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify possible molecular targets of PTP1B that mediate its positive role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify the Cyclin-dependent kinase 3 (Cdk3) as a novel PTP1B substrate. Molecular docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at Tyrosine residue 15 in vitro and interacts with it in the nucleus and cytoplasm of human glioblastoma (GB) cells. Finally, we found that the pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with the diminished activity of Cdk3, the consequent hypophosphorylation of Rb, and the down-regulation of E2F and its target genes Cdk1, Cyclin A, and Cyclin E1. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells and suggest new therapeutic strategies for treating these tumors.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Glioblastoma

SUBMITTER: Jonathan Chernoff

LAB HEAD: Jonathan Chernoff, MD, PhD

PROVIDER: PXD046000 | Pride | 2023-12-05

REPOSITORIES: Pride

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Project description:The retinoblastoma tumor suppressor protein (Rb) regulates early G1 phase checkpoints, including the DNA damage response, as well as cell cycle exit and differentiation. The widely accepted model of G1 cell cycle progression proposes that cyclin D:Cdk4/6 partially inactivates the Rb tumor suppressor during early G1 phase by progressive multi-phosphorylation, termed hypo-phosphorylation, resulting in release of E2F transcription factors. However, this model remains largely unproven biochemically and the biologically active form(s) of Rb remains unknown. Here we find that Rb is un-phosphorylated in G0 cells and becomes exclusively mono-phosphorylated throughout all of early G1 phase by cyclin D:Cdk4/6. Early G1 phase mono-phosphorylated Rb is composed of 14 independent isoforms that are all targeted by the E1a oncoprotein, but each shows a preferential binding pattern to specific E2F1-4 transcription factors. At the late G1 Restriction Point, cyclin E:Cdk2 inactivates Rb by a quantum hyper-phosphorylation (>12 phosphates/Rb). Cells undergoing a DNA damage response activate cyclin D:Cdk4/6 to generate mono-phosphorylated Rb that regulates global transcription. In contrast, a non-phosphorylatable ?Cdk-Rb allele was non-functional for regulating a DNA damage response, but functional for driving cell cycle exit and differentiation during myogenesis. These observations fundamentally change our understanding of G1 cell cycle progression and show that there is no progressive multi-phosphorylation or hypo-phosphorylation inactivation of Rb during early G1 phase by cyclin D:Cdk4/6. Instead, cyclin D:Cdk4/6 generates functionally active, mono-phosphorylated Rb that is the only Rb isoform present in cells during early G1 phase. Global transcriptional analysis of murine embryonic fibroblasts (MEFs) with conditional deletion of the endogenous RB gene by treatment with cell permeable TAT-Cre. Comparison to unaltered MEFs and MEFs with physiological level of exogenous wildtype or phospho-mutant RB expressed at time of RB gene deletion.

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A PTPB1-Cdk3 signaling axis promotes cell cycle progression of human glioblastoma cells through an Rb-E2F dependent pathway

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