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1. Alpha‐arrestins Aly1/Art6 and Aly2/Art3 regulate trafficking of the glycerophosphoinositol transporter Git1 and impact phospholipid homeostasis

   https://doi.org/10.1111/boc.202100007  

   Robinson, Benjamin P. ; Hawbaker, Sarah ; Chiang, Annette ; Jordahl, Eric M. ; Anaokar, Sanket ; Nikiforov, Alexiy ; Bowman, II, Ray W. ; Ziegler, Philip ; McAtee, Ceara K. ; Patton‐Vogt, Jana ; et al ( October 2021 , Biology of the Cell)

   Phosphatidylinositol (PI) is an essential phospholipid, critical to membrane bilayers. The complete deacylation of PI by B‐type phospholipases produces intracellular and extracellular glycerophosphoinositol (GPI). Extracellular GPI is transported into the cell via Git1, a member of the Major Facilitator Superfamily of transporters at the yeast plasma membrane. Internalized GPI is degraded to produce inositol, phosphate and glycerol, thereby contributing to these pools.GIT1gene expression is controlled by nutrient balance, with phosphate or inositol starvation increasingGIT1expression to stimulate GPI uptake. However, less is known about control of Git1 protein levels or localization.

   We find that the α‐arrestins, an important class of protein trafficking adaptor, regulate Git1 localization and this is dependent upon their interaction with the ubiquitin ligase Rsp5. Specifically, α‐arrestin Aly2 stimulates Git1 trafficking to the vacuole under basal conditions, but in response to GPI‐treatment, either Aly1 or Aly2 promote Git1 vacuole trafficking. Cell surface retention of Git1, as occurs inaly1∆ aly2∆ cells, is linked to impaired growth in the presence of exogenous GPI and results in increased uptake of radiolabeled GPI, suggesting that accumulation of GPI somehow causes cellular toxicity. Regulation of α‐arrestin Aly1 by the protein phosphatase calcineurin improves steady‐state and substrate‐induced trafficking of Git1, however, calcineurin plays a larger role in Git1 trafficking beyond regulation of α‐arrestins. Interestingly, loss of Aly1 and Aly2 increased phosphatidylinositol‐3‐phosphate on the limiting membrane of the vacuole, and this was further exacerbated by GPI addition, suggesting that the effect is partially linked to Git1. Loss of Aly1 and Aly2 leads to increased incorporation of inositol label from [³H]‐inositol‐labelled GPI into PI, confirming that internalized GPI influences PI balance and indicating a role for the a‐arrestins in this regulation.

   The α‐arrestins Aly1 and Aly2 are novel regulators of Git1 trafficking with previously unanticipated roles in controlling phospholipid distribution and balance.

   To our knowledge, this is the first example of α‐arrestin regulation of phosphatidyliniositol‐3‐phosphate levels. In future studies it will be exciting to determine if other α‐arrestins similarly alter PI and PIPs to change the cellular landscape.

    

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2. A Ubiquitously Conserved Cyanobacterial Protein Phosphatase Essential for High Light Tolerance in a Fast-Growing Cyanobacterium

   https://doi.org/10.1128/spectrum.01008-22  

   Walker, Patricia L. ; Pakrasi, Himadri B. ( August 2022 , Microbiology Spectrum)

   Youssef, Noha H. (Ed.)

   ABSTRACT Synechococcus elongatus UTEX 2973, the fastest-growing cyanobacterial strain known, optimally grows under extreme high light (HL) intensities of 1,500–2,500 μmol photons m −2 s −1 , which is lethal to most other photosynthetic microbes. We leveraged the few genetic differences between Synechococcus 2973 and the HL sensitive strain Synechococcus elongatus PCC 7942 to unravel factors essential for the high light tolerance. We identified a novel protein in Synechococcus 2973 that we have termed HltA for H igh l ight t olerance protein A . Using bioinformatic tools, we determined that HltA contains a functional PP2C-type protein phosphatase domain. Phylogenetic analysis showed that the PP2C domain belongs to the bacterial-specific Group II family and is closely related to the environmental stress response phosphatase RsbU. Additionally, we showed that unlike any previously described phosphatases, HltA contains a single N-terminal regulatory GAF domain. We found hltA to be ubiquitous throughout cyanobacteria, indicative of its potentially important role in the photosynthetic lifestyle of these oxygenic phototrophs. Mutations in the hltA gene resulted in severe defects specific to high light growth. These results provide evidence that hltA is a key factor in the tolerance of Synechococcus 2973 to high light and will open new insights into the mechanisms of cyanobacterial light stress response. IMPORTANCE Cyanobacteria are a diverse group of photosynthetic prokaryotes. The cyanobacterium Synechococcus 2973 is a high light tolerant strain with industrial promise due to its fast growth under high light conditions and the availability of genetic modification tools. Currently, little is known about the high light tolerance mechanisms of Synechococcus 2973, and there are many unknowns overall regarding high light tolerance of cyanobacteria. In this study, a comparative genomic analysis of Synechococcus 2973 identified a single nucleotide polymorphism in a locus encoding a serine phosphatase as a key factor for high light tolerance. This novel GAF-containing phosphatase was found to be the sole Group II metal-dependent protein phosphatase that is evolutionarily conserved throughout cyanobacteria. These results shed new light on the light response mechanisms of Synechococcus 2973, improving our understanding of environmental stress response. Additionally, this work will help facilitate the development of Synechococcus 2973 as an industrially useful organism. 

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3. The flip side of phospho‐signalling: Regulation of protein dephosphorylation and the protein phosphatase 2Cs

   https://doi.org/10.1111/pce.13616  

   Bhaskara, Govinal Badiger ; Wong, Min May ; Verslues, Paul E. ( August 2019 , Plant, Cell & Environment)

   Protein phosphorylation is a key signalling mechanism and has myriad effects on protein function. Phosphorylation by protein kinases can be reversed by protein phosphatases, thus allowing dynamic control of protein phosphorylation. Although this may suggest a straightforward kinase–phosphatase relationship, plant genomes contain five times more kinases than phosphatases. Here, we examine phospho‐signalling from a protein phosphatase centred perspective and ask how relatively few phosphatases regulate many phosphorylation sites. The most abundant class of plant phosphatases, the protein phosphatase 2Cs (PP2Cs), is surrounded by a web of regulation including inhibitor and activator proteins as well as posttranslational modifications that regulate phosphatase activity, control phosphatase stability, or determine the subcellular locations where the phosphatase is present and active. These mechanisms are best established for the Clade A PP2Cs, which are key components of stress and abscisic acid signalling. We also describe other PP2C clades and illustrate how these phosphatases are highly regulated and involved in a wide range of physiological functions. Together, these examples of multiple layers of phosphatase regulation help explain the unbalanced kinase–phosphatase ratio. Continued use of phosphoproteomics to examine phosphatase targets and phosphatase–kinase relationships will be important for deeper understanding of phosphoproteome regulation.

    

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4. Pseudophosphatases as Regulators of MAPK Signaling

   https://doi.org/10.3390/ijms222212595  

   Hepworth, Emma Marie ; Hinton, Shantá D. ( November 2021 , International Journal of Molecular Sciences)

   Mitogen-activated protein kinase (MAPK) signaling pathways are highly conserved regulators of eukaryotic cell function. These enzymes regulate many biological processes, including the cell cycle, apoptosis, differentiation, protein biosynthesis, and oncogenesis; therefore, tight control of the activity of MAPK is critical. Kinases and phosphatases are well established as MAPK activators and inhibitors, respectively. Kinases phosphorylate MAPKs, initiating and controlling the amplitude of the activation. In contrast, MAPK phosphatases (MKPs) dephosphorylate MAPKs, downregulating and controlling the duration of the signal. In addition, within the past decade, pseudoenzymes of these two families, pseudokinases and pseudophosphatases, have emerged as bona fide signaling regulators. This review discusses the role of pseudophosphatases in MAPK signaling, highlighting the function of phosphoserine/threonine/tyrosine-interacting protein (STYX) and TAK1-binding protein (TAB 1) in regulating MAPKs. Finally, a new paradigm is considered for this well-studied cellular pathway, and signal transduction pathways in general. 

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5. Promoting the activity of a receptor tyrosine phosphatase with a novel pH ‐responsive transmembrane agonist inhibits cancer‐associated phenotypes

   https://doi.org/10.1002/pro.4742  

   Rizzo, Sophie ; Sikorski, Eden ; Park, Soohyung ; Im, Wonpil ; Vasquez‐Montes, Victor ; Ladokhin, Alexey S. ; Thévenin, Damien ( August 2023 , Protein Science)

   Cell signaling by receptor protein tyrosine kinases (RTKs) is tightly controlled by the counterbalancing actions of receptor protein tyrosine phosphatases (RPTPs). Due to their role in attenuating the signal‐initiating potency of RTKs, RPTPs have long been viewed as therapeutic targets. However, the development of activators of RPTPs has remained limited. We previously reported that the homodimerization of a representative member of the RPTP family (protein tyrosine phosphatase receptor J or PTPRJ) is regulated by specific transmembrane (TM) residues. Disrupting this interaction by single point mutations promotes PTPRJ access to its RTK substrates (e.g., EGFR and FLT3), reduces RTK's phosphorylation and downstream signaling, and ultimately antagonizes RTK‐driven cell phenotypes. Here, we designed and tested a series of first‐in‐class pH‐responsive TM peptide agonists of PTPRJ that are soluble in aqueous solution but insert as a helical TM domain in lipid membranes when the pH is lowered to match that of the acidic microenvironment of tumors. The most promising peptide reduced EGFR's phosphorylation and inhibited cancer cell EGFR‐driven migration and proliferation, similar to the PTPRJ's TM point mutations. Developing tumor‐selective and TM‐targeting peptide binders of critical RPTPs could afford a potentially transformative approach to studying RPTP's selectivity mechanism without requiring less specific inhibitors and represent a novel class of therapeutics against RTK‐driven cancers.

    

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