Phosphatases are a diverse family of enzymes, comprising at least 10 distinct protein folds. Like most other enzyme families, many have sequence variations that predict an impairment or loss of catalytic activity classifying them as pseudophosphatases. Research on pseudoenzymes is an emerging area of interest, with new biological functions repurposed from catalytically active relatives. Here, we provide an overview of the pseudophosphatases identified to date in all major phosphatase families. We will highlight the degeneration of the various catalytic sequence motifs and discuss the challenges associated with the experimental determination of catalytic inactivity. We will also summarize the role of pseudophosphatases in various diseases and discuss the major challenges and future directions in this field.

LITAF is a small cellular protein with an unknown function. The C-terminus of LITAF contains a highly conserved domain termed the SIMPLE-like domain (SLD), while the N-terminus contains two PPXY motifs that mediate protein-protein interactions with WW-domain containing proteins. LITAF also harbors two endosome/lysosome targeting sequences at its C-terminus, but there has been conflicting reports regarding its intracellular localization. Here, we demonstrate that LITAF is localized to the late endosome/lysosomal compartment in a variety of cell lines. We also show that Itch, a WW-domain containing protein, and LITAF strongly interact and that this interaction depends on the two PPXY motifs in the N-terminus of LITAF. Interestingly, co-expression of LITAF with Itch induces major changes in Itch intracellular localization, bringing Itch from the trans-Golgi network to lysosomes. We show that this re-localization is dependent upon the interaction with the PPXY sequences of LITAF, since disruption of these binding motifs completely abrogates Itch re-localization.

The truncated C‐terminal portion of Bid (tBid) is an important intermediate in ligand‐induced apoptosis. tBid has been shown to be sensitive to proteasomal inhibitors and downregulated by activation of the epidermal growth factor (EGF) pathway. Here, we provide evidence that tBid is a substrate of the ubiquitin ligase Itch, which can specifically interact with and ubiquitinate tBid, but not intact Bid. Consistently, overexpression of Itch increases cell survival and inhibits caspase 3 activity, whereas downregulation of Itch by RNA interference has the opposite effect, increasing cell death and apoptosis. Treatment with EGF increases Itch phosphorylation and activity, and Itch expression is important for the ability of EGF to increase cell survival after tumour necrosis factor‐related apoptosis‐inducing ligand treatment. Our findings identify Itch as a key molecule between EGF signalling and resistance to apoptosis through downregulation of tBid, providing further details on how EGF receptor and proteasome inhibitors can contribute to the induction of apoptosis and the treatment of cancer. Structural digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7542954: ITCH (uniprotkb:http://www.ebi.uniprot.org/entry/Q96J02) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with tBid (uniprotkb:http://www.ebi.uniprot.org/entry/P70444) by anti tag coimmunoprecipitation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7542970: tBid (uniprotkb:http://www.ebi.uniprot.org/entry/P70444) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with Ubiquitin (uniprotkb:http://www.ebi.uniprot.org/entry/P62988) by anti tag coimmunoprecipitation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7542986: ITCH (uniprotkb:http://www.ebi.uniprot.org/entry/Q96J02) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with tBid (uniprotkb:http://www.ebi.uniprot.org/entry/P70444) by bioluminescence resonance energy transfer (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0012)

Itch is a member of the C2-WW-HECT (CWH) family of ubiquitin ligases involved in the control of inflammatory signaling pathways, several transcription factors, and sorting of surface receptors to the degradative pathway. In addition to these common domains, Itch also contains a conserved proline-rich region (PRR) allowing its interaction with Src homology 3 (SH3) domain-containing proteins. This region is composed of 20 amino acids and contains one consensus class I and three class II SH3-binding motifs. Several SH3 domain-containing partners have been shown to recognize the Itch PRR, but their binding properties have been poorly defined. Here we compare a subset of endocytic SH3 domain-containing proteins using bioluminescence resonance energy transfer, isothermal titration calorimetry, and pull-down assays. Results indicate that Endophilin is a high-affinity binding partner of Itch both in vivo and in vitro, with a calculated KD placing this complex among the highest-affinity SH3 domain-mediated interactions reported to date. All of the SH3 domains tested here bind to Itch with a 1:1 stoichiometry, except for β-PIX that binds with a 2:1 stoichiometry. Together, these results indicate that Itch PRR is a versatile binding module that can accommodate several different SH3 domain-containing proteins but has a preference for Endophilin. Interestingly, the catalytic activity of Itch toward different SH3 domain-containing proteins was similar, except for β-PIX that was not readily ubiquitylated even though it could interact with an affinity comparable to those of other substrates tested.