The pseudophosphatases, atypical members of the protein tyrosine phosphatase family, have emerged as bona fide signaling regulators within the past two decades. Their roles as regulators have led to a renaissance of the pseudophosphatase and pseudoenyme fields, catapulting interest from a mere curiosity to intriguing and relevant proteins to investigate. Pseudophosphatases make up approximately fourteen percent of the phosphatase family, and are conserved throughout evolution. Pseudophosphatases, along with pseudokinases, are important players in physiology and pathophysiology. These atypical members of the protein tyrosine phosphatase and protein tyrosine kinase superfamily, respectively, are rendered catalytically inactive through mutations within their catalytic active signature motif and/or other important domains required for catalysis. This new interest in the pursuit of the relevant functions of these proteins has resulted in an elucidation of their roles in signaling cascades and diseases. There is a rapid accumulation of knowledge of diseases linked to their dysregulation, such as neuropathies and various cancers. This review analyzes the involvement of pseudophosphatases in diseases, highlighting the function of various role(s) of pseudophosphatases involvement in pathologies, and thus providing a platform to strongly consider them as key therapeutic drug targets.

The catalytically inactive mitogen-activated protein (MAP) kinase phosphatase, MK-STYX (MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein) interacts with the stress granule nucleator G3BP-1 (Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1), and decreases stress granule (stalled mRNA) formation. Histone deacetylase isoform 6 (HDAC6) also binds G3BP-1 and serves as a major component of stress granules. The discovery that MK-STYX and HDAC6 both interact with G3BP-1 led us to investigate the effects of MK-STYX on HDAC6 dynamics. In control HEK/293 cells, HDAC6 was cytosolic, as expected, and formed aggregates under conditions of stress. In contrast, in cells overexpressing MK-STYX, HDAC6 was both nuclear and cytosolic and the number of stress-induced aggregates significantly decreased. Immunoblots showed that MK-STYX decreases HDAC6 serine phosphorylation, protein tyrosine phosphorylation, and lysine acetylation. HDAC6 is known to regulate microtubule dynamics to form aggregates. MK-STYX did not affect the organization of microtubules, but did affect their post-translational modification. Tubulin acetylation was increased in the presence of MK-STYX. In addition, the detyrosination of tubulin was significantly increased in the presence of MK-STYX. These findings show that MK-STYX decreases the number of HDAC6-containing aggregates and alters their localization, sustains microtubule acetylation, and increases detyrosination of microtubules, implicating MK-STYX as a signaling molecule in HDAC6 activity.

Macroautophagy (autophagy) is a critical process that degrades cytoplasmic components to maintain homeostasis and which requires microtubule interactions to function. The autophagosomes (double membrane vesicles) envelop substrates and then are transported along microtubules through motor proteins to fuse with degradative lysosomes. The formation of the autolysosome allows the breakdown and degradation of various cellular targets such as stress granules (SG; stalled mRNA). We reported that the pseudophosphatase MK‐STYX [MAPK (mitogen‐activated protein kinase) phosphoserine/threonine/tyrosine‐binding protein] inhibits SG. Since autophagy and MK‐STYX each negatively affect stress granule assembly, we sought to determine whether MK‐STYX has a role in autophagy and microtubule dynamics. Our studies show that MK‐STYX causes cytosolic TFEB (Transcription factor E‐Box; the autophagy “master switch”) to become nuclear and form peripheral nuclear aggregates independently of nutrient status. Whereas, MK‐STYXactive (active mutant in which catalytic activity has been “restored”), only increased TFEB cytosolic aggregates in serum starved cells. TFEB also localizes to the lysosomal surface, acting as negative regulator of lysosomal and autophagosomal biogenesis. MK‐STYX alters lysosomal and autophagosomal dynamics; we observed oversized lysosomes in the presence of MK‐STYX. Furthermore, autophagosomes localized to the distal ends of HEK‐293 cellular extensions in the presence of MK‐STYX. In addition, MK‐STYX increases the phosphorylation of the essential autophagosome constituent LC3 (microtubule‐associated proteins 1A/1B light chain 3B). To determine MK‐STYX's effects on microtubule function, we over‐expressed MK‐STYX in HEK‐293 cells in the presence or absence of nocodazole, which disrupts microtubules. MK‐STYX did not affect microtubules dynamics directly, suggesting that further analysis of the motor proteins involved in stress granule regulation and/or autophagy is needed. Investigating MK‐STYX as a player in autophagic mechanisms is an intriguing scientific trajectory. Autophagy plays a major role in prominent cellular processes ranging from initial developmental stages to the onset of progressive human pathologies such as neurodegenerative diseases and cancer. These studies suggest that MK‐STYX may have an important role in autophagy, illuminating the importance of pseudoenzymes in regulating critical cellular pathways.Support or Funding InformationSupport for this research was provided by two Howard Hughes Medical Institute Research Fellowships and a National Science Foundation Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.