Biology subjects

Greiner, E. C.

Publications and source records attributed to Greiner, E. C..

3 recordsLinked to original sources

bioRxiv2025

A functional map of phosphoprotein phosphatase regulation identifies an evolutionary conserved reductase for the catalytic metal ions

Serine/Threonine phosphoprotein phosphatases (PPPs, PP1-PP7) are conserved metalloenzymes and central to intracellular signaling in eukaryotes, but the details of their regulation is poorly understood. To address this, we performed genome-wide CRISPR knockout and focused base editor screens in PPP perturbed conditions to establish a high-resolution functional map of PPP regulation that pinpoints novel regulatory mechanisms. Through this, we identify the orphan reductase CYB5R4 as an evolutionarily conserved activator of PP4 and PP6, but not the closely related PP2A. Heme binding is essential for CYB5R4 function and mechanistically involves the reduction of the metal ions in the active site. Importantly, CYB5R4-mediated activation of PP4 is critical for cell viability when cells are treated with DNA damage-inducing agents known to cause oxidative stress. The discovery of a dedicated PPP reductase points to shared regulatory principles with protein tyrosine phosphatases, where specific enzymes dictate activity by regulating the active site redox state. In sum, our work provides a resource for understanding PPP function and the regulation of intracellular signaling.

cell biology↗

bioRxiv2024

PLK-1 regulates MEX-1 polarization in the C. elegans zygote

The one-cell C. elegans embryo undergoes an asymmetric cell division during which germline factors such as the RNA-binding proteins POS-1 and MEX-1 segregate to the posterior cytoplasm, leading to their asymmetric inheritance to the posterior germline daughter cell. Previous studies found that the RNA-binding protein MEX-5 recruits polo-like kinase PLK-1 to the anterior cytoplasm where PLK-1 inhibits the retention of its substrate POS-1, leading to POS-1 segregation to the posterior. In this study, we tested whether PLK-1 similarly regulates MEX-1 polarization. We find that both the retention of MEX-1 in the anterior and the segregation of MEX-1 to the posterior depend on PLK kinase activity and on the interaction between MEX-5 and PLK-1. Human PLK1 directly phosphorylates recombinant MEX-1 on 9 predicted PLK-1 sites in vitro, four of which were identified in previous phosphoproteomic analysis of C. elegans embryos. The introduction of alanine substitutions at these four PLK-1 phosphorylation sites (MEX-1(4A)) significantly weakened the inhibition of MEX-1 retention in the anterior, thereby weakening MEX-1 segregation to the posterior. In contrast, mutation of a predicted CDK1 phosphorylation site had no effect on MEX-1 retention or on MEX-1 segregation. MEX-1(4A) mutants are viable and fertile but display significant sterility and fecundity defects at elevated temperatures. Taken together with our previous findings, these findings suggest PLK-1 phosphorylation drives both MEX-1 and POS-1 polarization during the asymmetric division of the zygote.

developmental biology↗

bioRxiv2021

Regulation of PP2A, PP4, and PP6 holoenzyme assembly by carboxyl-terminal methylation

The family of Phosphoprotein Phosphatases (PPPs) is responsible for most cellular serine and threonine dephosphorylation. PPPs achieve substrate specificity and selectivity by forming multimeric holoenzymes. PPP holoenzyme assembly is tightly controlled, and changes in the cellular repertoire of PPPs are linked to human disease, including cancer and neurodegeneration. For PP2A, PP4, and PP6, holoenzyme formation is in part regulated by carboxyl (C)-terminal methyl-esterification (often referred to as "methylation"). Here, we use mass spectrometry-based proteomics, methylation-ablating mutations, and genome editing to elucidate the role of C-terminal methylation on PP2A, PP4, and PP6 holoenzyme assembly. We find that the catalytic subunits of PP2A, PP4, and PP6 are frequently methylated in cancer cells and that deletion of the C-terminal leucine faithfully recapitulates loss of methylation. We observe that loss of PP2A methylation consistently reduced B55, B56, and B72 regulatory subunit binding in cancer and non-transformed cell lines. However, Striatin subunit binding is only affected in non-transformed cells. For PP4, we find that PP4R1 and PP4R3{beta} bind in a methylation-dependent manner. Intriguingly, loss of methylation does not affect PP6 holoenzymes. Our analyses demonstrate in an unbiased, comprehensive, and isoform-specific manner the crucial regulatory function of endogenous PPP methylation in transformed and non-transformed cell lines

biochemistry↗