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Quantitative phosphoproteomics of Mycolicibacterium smegmatis

ABSTRACT: Protein phosphorylation plays versatile and important roles in the pathogen and host physiology in Mycobacterium tuberculosis, which holding promise as new targets for anti-tuberculosis drugs. Mycolicibacterium smegmatis was used as a model organism to study the protein phosphorylation at different growth phases because of its close association with M.tuberculosis. We identified 573 phosphorylated peptides and 933 phosphorylation sites from 385 proteins in samples from both logarithmic and stationary phase of M.smegmatis. The number of identified phosphorylation sites is 4.2 times higher than the largest phosphoproteome discovered to date, establishing a comprehensive dataset of phosphorylated proteins in M.smegmatis. By comparing the significantly differentially expressed phosphorylated proteins between the logarithmic phase and the stationary phase, we discovered 68 upregulated proteins involved in cell elongation and division pathways and 69 downregulated proteins involved in signaling pathways such as the tricarboxylic acid cycle. Differentially expressed proteins were found to regulate important cellular cycle events such as cell growth and proliferation, providing proteome level support for the elucidation of phosphorylation functions in both M. smegmatis and M. tuberculosis.

ORGANISM(S): Mycolicibacterium Smegmatis Mc2 155

SUBMITTER: Yao Zhang

PROVIDER: PXD052425 | iProX | Mon May 20 00:00:00 BST 2024

REPOSITORIES: iProX

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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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Quantitative phosphoproteomics of Mycolicibacterium smegmatis

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