PubMed · 7499357
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A C Newton. 1995-12-01. Protein kinase C: structure, function, and regulation.. https://doi.org/10.1074/jbc.270.48.28495
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The Heterochromatin Protein 1 (HP1) family proteins are key regulators of chromatin structure and genome function, acting as "reader" proteins that recognize and bind to histone H3 lysine 9 methylation (H3K9me). Beyond their canonical role in heterochromatin formation and transcriptional repression, HP1 proteins exhibit functional versatility, participating in transcriptional activation, RNA processing, DNA repair, and chromosome segregation. This multifunctionality is mediated partially by post-translational modifications (PTMs), with phosphorylation emerging as a central regulatory mechanism. This review explores the diverse effects of HP1 phosphorylation on protein function and chromatin interactions, focusing on Drosophila melanogaster HP1a and its orthologs, mammalian HP1α and S. pombe Swi6. Phosphorylation in the N-terminal tail enhances HP1's affinity for H3K9me, promoting transcriptional silencing. Mitotic phosphorylation of serine residues in the hinge region, regulated by kinases such as AURKB and NDR1/2, leads to chromatin release and relocalization to the kinetochore, enabling proper chromosome segregation. Additionally, phosphorylation modulates HP1 phase separation dynamics, influencing nuclear compartmentalization and chromatin condensation. These findings highlight phosphorylation as a versatile molecular switch that enables HP1 proteins to transition between structural and regulatory roles, contributing to their evolutionary conserved multifunctionality in genome regulation and cell division. Further investigation into HP1 phosphorylation across species and contexts is essential to fully understand its contributions to chromatin biology.
Cell survival depends upon the ability to adapt to changing environments. Environmental stressors trigger an acute stress response program that rewires cell physiology, downregulates proliferation genes and pauses the cell cycle until the cell adapts. After the acute response is resolved, cells resume cycling but at a reduced rate. The importance of cell cycle changes for survival in chronic stress is not clear. Here, we show that dynamic phosphorylation of the yeast cell cycle-regulatory transcription factor Hcm1 is required to maintain fitness in chronic stress. Hcm1 is activated by cyclin dependent kinase (CDK) during S-phase and is inactivated by the phosphatase calcineurin (CN) in response to stressors that signal through increases in cytosolic Ca2+. Cells expressing a constitutively active, phosphomimetic Hcm1 mutant exhibit a reduction in fitness in stress, suggesting Hcm1 inactivation promotes survival. However, a comprehensive analysis of Hcm1 phosphomutants revealed that Hcm1 activity is also important to survive stress, and that all mutants with fixed phosphorylation states are less fit in stress. Moreover, our data suggests that pulses of Hcm1 activity are necessary to maximize target gene expression in stress. These findings demonstrate that expression levels of Hcm1 target genes influence fitness in stress and suggest that the dynamic phosphorylation of cell cycle regulators plays a crucial role in promoting survival in stressful environments.
Protein phosphorylation stoichiometry was assessed by two analytical strategies. Both are based on element mass spectrometry (ICPMS, inductively coupled plasma mass spectrometry) and simultaneous monitoring of (31)P and (34)S. One strategy employs a combination of 1D gel electrophoresis, in-gel digestion, and final microLC-ICPMS analysis (microLC = capillary liquid chromatography). The other strategy uses the combination of 1D gel electrophoresis, protein blotting, and imLA-ICPMS (imLA = imaging laser ablation). The two methods were evaluated with standard phosphoproteins and were applied to the analysis of the cytoplasmatic proteome of bacterial cells (Corynebacterium glutamicum) and eukaryotic cells (Mus musculus). The eukaryotic proteome was found to exhibit a significantly higher phosphorylation degree (approximately 0.8 mol of P/mol of protein) compared to the bacterial proteome (approximately 0.01 mol of P/mol of protein). Both analytical strategies revealed consistent quantitative results, with the microLC-ICPMS approach providing the higher sensitivity. In summary, two ICPMS-based methods for quantitative estimation of the phosphorylation degree of a cellular proteome are presented which access the native proteome state and do not require any type of label introduction or derivatization.