Specific substrate recruitment by the protein phosphatase-1 molecule G subunit holoenzyme enables the maintenance of glucose levels.

Polo-like kinase 1 (Plk1) is an essential protein kinase that promotes faithful mitotic progression in eukaryotes. The subcellular localization and substrate interactions of Plk1 are tightly controlled and require its binding to phosphorylated sequences. Here, to identify phosphorylation-dependent interactions within the Plk1 network in human mitotic cells we performed quantitative proteomics on HeLa cells cultured with kinase inhibitors or expressing a Plk1 mutant that was deficient in phosphorylation-dependent substrate binding. We found that many interactions were abolished upon kinase inhibition; however, a subset were protected from phosphatase opposition or were unopposed, resulting in persistent interaction of the substrate with Plk1. This subset includes phosphoprotein phosphatase 6 (PP6), whose activity towards Aurora kinase A (Aurora A) was inhibited by Plk1. Our data suggest that this Plk1-PP6 interaction creates a feedback loop that coordinates and reinforces the activities of Plk1 and Aurora A during mitotic entry and is terminated by the degradation of Plk1 during mitotic exit. Thus, we have identified a mechanism for the previously puzzling observation of Plk1-dependent regulation of Aurora A.

A "tug-of-war" between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases.We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the "PPPome"). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.

It is unknown how cues from the tumor microenvironment can regulate post-transcriptional mechanisms, such as alternative splicing, that control genes that drive malignant growth. The induction of cyclooxygenase 2 (Cox-2) by integrin a3b1 in breast cancer cells can promote tumor progression. We have used RNAi to suppress a3b1 in human MDA-MB-231 breast cancer cells and then investigated changes in global gene expression. Numerous mRNAs, including Cox-2, show altered expression and/or alternative exon usage (AEU) in a3b1-deficient cells. AEU included patterns predicted to render an mRNA susceptible to degradation, such as 39-UTR variations or retention of elements that target an mRNA for nonsense-mediated decay (NMD). PCR-based analysis of a3b1-deficient cells confirmed changes in Cox-2 mRNA that might target it for NMD, including retention of an intron that harbors premature termination codons and changes within the 39-UTR. Moreover, Cox-2 mRNA has reduced stability in a3b1-deficient cells, which is partially reversed by knockdown of the essential NMD factor UPF1. Our study identifies a3b1-mediated AEU as a novel paradigm of integrin-dependent gene regulation that has potential for exploitation as a therapeutic target.

The development of novel therapies to promote wound healing is hindered by our poor understanding of how different integrins function together in the epidermis. Longmate et al. show that cross-suppression by integrins within the epidermis controls paracrine signals that regulate wound angiogenesis. Integrin α9β1 suppresses the proangiogenic functions of α3β1 during late-stage wound healing, leading to the normalization of blood vessel density in the wound bed.

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 PP4R3β 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.

Proteolytic processing of the laminin-γ2 chain is a hallmark of basement membrane maturation in the skin. Integrin α3β1, a major receptor for epidermal adhesion to laminin-332, is critical for proper basement membrane organization during skin development and wound healing. Previously, we identified a role for α3β1 in promoting the processing of laminin-γ2 in cultured keratinocytes in vitro and in wound epidermis in vivo. In this study we identify the Bmp1 gene, which encodes variants of the mTLD/BMP-1 metalloproteases, as a critical regulator of α3β1-dependent laminin-γ2 processing, thereby expanding the role of this integrin in controlling the secretion by the epidermis of factors that modulate the tissue microenvironment. Because our previous studies identified another epidermal integrin, α9β1, as a suppressive regulator of α3β1-dependent wound angiogenesis, we investigated whether α9β1 has a similar cross-suppressive effect on the ability of α3β1 to promote basement membrane organization. Here, we show that, rather than a cross-suppressive role, α9β1 has an opposing role in basement membrane assembly/maturation through reduced laminin-γ2 processing via mTLD/BMP-1. Although α3β1 promotes this process during wound healing, α9β1 has an inhibitory role, suggesting that regulation of basement membrane assembly requires a complex interplay between these distinct epidermal integrins.

Objective Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane endoplasmic/sarcoplasmic reticulum (E/SR) protein recognized for its role in a store operated Ca 2+ entry (SOCE), an ancient and ubiquitous signaling pathway. Whereas STIM1 is known to be indispensable during development, its biological and metabolic functions in mature muscles remain unclear. Methods Conditional and tamoxifen inducible muscle STIM1 knock-out mouse models were coupled with multi-omics tools and comprehensive physiology to understand the role of STIM1 in regulating SOCE, mitochondrial quality and bioenergetics, and whole-body energy homeostasis. Results This study shows that STIM1 is abundant in adult skeletal muscle, upregulated by exercise, and is present at SR-mitochondria interfaces. Inducible tissue-specific deletion of STIM1 (iSTIM1 KO) in adult muscle led to diminished lean mass, reduced exercise capacity, and perturbed fuel selection in the settings of energetic stress, without affecting whole-body glucose tolerance. Proteomics and phospho-proteomics analyses of iSTIM1 KO muscles revealed molecular signatures of low-grade E/SR stress and broad activation of processes and signaling networks involved in proteostasis. Conclusion These results show that STIM1 regulates cellular and mitochondrial Ca 2+ dynamics, energy metabolism and proteostasis in adult skeletal muscles. Furthermore, these findings provide insight into the pathophysiology of muscle diseases linked to disturbances in STIM1-dependent Ca 2+ handling.