TULA proteins as signaling regulators

Journal Pre-proof TULA PROTEINS AS SIGNALING REGULATORS Alexander Y. Tsygankov

PII:

S0898-6568(19)30220-7

DOI:

https://doi.org/10.1016/j.cellsig.2019.109424

Reference:

CLS 109424

To appear in: Received Date:

29 August 2019

Revised Date:

16 September 2019

Accepted Date:

18 September 2019

Please cite this article as: Tsygankov AY, TULA PROTEINS AS SIGNALING REGULATORS, Cellular Signalling (2019), doi: https://doi.org/10.1016/j.cellsig.2019.109424

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TULA PROTEINS AS SIGNALING REGULATORS

Alexander Y. Tsygankov

Sol Sherry Thrombosis Research Center, Fels Institute for Cancer Research and Department of Microbiology and Immunology, Lewis Katz School of Medicine at Temple

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University, 3400 N. Broad Street, Philadelphia, PA 19140; phone: 215-707-1745; email:

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[email protected]

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HIGHLIGHTS

UBASH3/STS/TULA-family proteins consist of the UBA, SH3 and phosphatase

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domains

The two family members markedly differ in tissue expression and phosphatase

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activity

Regulatory effects of these proteins mostly depend on their phosphatase activity

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TULA proteins regulate signaling in various systems, including T cells and

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platelets

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Down-regulation of Syk-mediated signaling is to-date the best-studied effect of

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TULA-2

ABSTRACT

Two members of the UBASH3/STS/TULA family exhibit a unique protein domain structure, which includes a histidine phosphatase domain, and play a key role in regulating cellular signaling. UBASH3A/STS-2/TULA is mostly a lymphoid protein, while UBASH3B/STS-1/TULA-2 is expressed ubiquitously. Dephosphorylation of tyrosine-

phosphorylated proteins by TULA-2 and, probably to a lesser extent, by TULA critically contribute to the molecular basis of their regulatory effect. The notable differences between the effects of the two family members on cellular signaling and activation are likely to be linked to the difference between their specific enzymatic activities. However, these differences might also be related to the functions of their domains other than the phosphatase domain and independent of their phosphatase activity. The down-

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regulation of the Syk/Zap-70-mediated signaling, which to-date appears to be the beststudied regulatory effect of TULA family, is discussed in detail in this publication.

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ABBREVIATIONS

AIF, apoptosis-inducing factor; AML, acute myeloid leukemia; CLEC-2; C-type lectin-like

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receptor 2; DC, dendritic cell; EGFR, epithelial growth factor receptor; FDP, fluorescein

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diphosphate; FLT3, Fms-like tyrosine kinase; SNP, single nucleotide polymorphisms; ITAM, immunoreceptor tyrosine-based activation motif; KO, knockout; miR, microRNA;

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OMFP, 3-O-methylfluorescein phosphate; pNPP, p-nitrophenyl phosphate; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; pY, phosphotyrosine; STS,

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suppressor of T-cell signaling; TCR, T-cell receptor; TULA, T-cell ubiquitin ligand; UBASH3, ubiquitin-associated and Src-homology 3; WT, wild-type KEYWORDS

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UBASH3A; UBASH3B; STS-1; STS-2; TULA; TULA-2

INTRODUCTION The two members of the UBASH3 family were discovered independently by

several research groups approximately 15 years ago 1-5. These genes/proteins are annotated in the relevant databases as UBASH3A and UBASH3B; UBASH3 stands for ubiquitin-associated (UBA) and Src-homology 3 (SH3) domain. Several synonyms are utilized to denote them; UBASH3A is called STS-2 for Suppressor of T-cell Signaling,

TULA for T-cell Ubiquitin Ligand (in some reports it is called TULA-1), and CLIP4 for CblInteracting Protein 4, whereas UBASH3B is called STS-1, TULA-2, and p70. The terms TULA and TULA-2 are mostly used in this publication exclusively for the sake of consistency with other publications of the author. Several reviews covering the studies have been published addressing the initial studies of the family, the structure and expression of the TULA proteins, the relationships between the family members in

STRUCTURE AND EXPRESSION OF TULA PROTEINS

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mostly focused on the role played by this family in cellular signaling.

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various taxonomic groups and their functions in health and disease 6-9. This review is

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The structure of TULA and TULA-2 includes the interactive domains UBA and

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SH3, and the histidine phosphatase enzymatic domain 10 (Fig. 1). The contribution of UBA 4,5,11-13 and SH3 4,5,14 domains to the functions of TULA and TULA-2 has been

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shown, although many questions related to their role remain. The histidine phosphatase domain, which is termed this way because of the presence of a key histidine residue in

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its active site capable of catalyzing phosphatase and, in some proteins, mutase reactions 10, is a key functional region of TULA-family proteins 3,15-17. The signaling functions of these proteins appear to be causally linked to their phosphatase activity toward various substances of which phosphotyrosine (pY)-containing proteins and peptides serve as their critical physiological substrates 15-19. The most C-terminal region

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of the TULA proteins mediates their dimerization 4,15,16. Although oligomerization is known to play a role in the enzymatic activity in general, the role of oligomerization in the functions of TULA family remains to be understood. Finally, the region between UBA and SH3 contains the domain corresponding to the 2H phosphoesterase superfamily, which includes several families with two conserved histidines in the active site capable of 2’,3’

cyclic nucleotide phosphodiesterase activity 20. The role of this putative domain and its activity in the context of the TULA family remains to be understood. Members of the UBASH3/STS/TULA family exist in most metazoan species with a very clear distinction between vertebrates and invertebrates: vertebrate species have two members (although there is a serious difference between bony fishes and the rest of the vertebrates, since the bony fishes have two family members similar to TULA-2 and

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none similar to TULA), while invertebrates, including chordates, have only one TULA-like protein, which exhibits similarity to both TULA and TULA-2 9. One important conclusion

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drawn from the comparison of TULA-family members from different species is that

TULA-2 is highly conserved, while TULA is not. Human TULA-2 shows 98, 90 and 82%

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identity, respectively, with TULA-2 from mouse, chicken and coelacanth (Latimeria

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chalumnae), a lobe-finned fish, presumably similar to the common ancestor of all tetrapodes. In stark contrast, human TULA shows 83, 67 and 55% identity with TULA of

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these species; notably, the degree of identity between human and mouse TULA corresponds to that between human and coelacanth TULA-2 9.

5,21-24,

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Expression of TULA and TULA-2 has been studied only in mice and humans 1and the initial conclusion of these studies is that TULA is a lymphoid-specific

protein, while TULA-2 is expressed ubiquitously 6-8. However, some findings indicate that their differential expression is not as clear-cut. Thus, the presence of TULA has been demonstrated in mouse mast cell lines 23 and human platelets, albeit at a level one-tenth

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of that for TULA-2 24. Furthermore, the expression levels of these proteins change in the course of cell differentiation. Thus, it has been shown that although both TULA and TULA-2 are expressed in fresh-isolated bone marrow cells, their in-vitro differentiation toward macrophages or dendritic cells (DC) suppresses TULA expression, while maintaining TULA-2 at the original level in macrophages or even up-regulating it in DCs 22.

Likewise, the level of TULA-2 is greatly increased along the route of megakaryocyte

differentiation (our unpublished data). Overall, the conclusion that TULA is mostly a lymphoid protein remains valid, but its expression in other cell types, albeit at a lower level, cannot be ruled out. The interest in this family is due not only to its unique domain architecture and the unusual nature of its phosphatase domain, but also to its biological functions. TULA and TULA-2 play a key regulatory role in the responses of various cell types in health

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and disease. Ample evidence links the activity of TULA and/or TULA-2 to the functions of T lymphocytes, platelets, macrophages, basophils, mast cells and other cells in vitro

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and in vivo 3,21-23,25-27. Furthermore, the links between sequence variability in the genes

encoding for TULA-family proteins and several diseases of autoimmune and/or chronic

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inflammatory nature have been established (reviewed in 8,9). This review will be referring

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to some of the biological functions of TULA-family proteins only when it is needed to demonstrate the link between these functions and the effects of these proteins on

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cellular signaling, which is the main focus of this review.

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CELLULAR EFFECTS OF TULA PROTEINS DEPEND ON THEIR PHOSPHATASE ACTIVITY

The up-regulation effect of TULA/TULA-2 (STS-2/STS-1) double knockout (dKO)

on tyrosine phosphorylation of the key T-cell protein tyrosine kinase (PTK) Zap-70 and,

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in general, on Zap-70-dependent signaling induced by the engagement of the T-cell receptor for antigen (TCR), which correlated well with a similar effect of dKO on T-cell proliferation and cytokine secretion was demonstrated in the initial report characterizing these mice. Notably, a strong synergism of the two single KO (sKO) has been shown, since the effect of either sKO on proliferation was negligible as compared to that of dKO 3,15.

Subsequently, it has been shown that TULA-2 (STS-1) has a protein tyrosine

phosphatase (PTP) activity, and that this activity is critical for the effect of TULA-2 on

signaling; when T cells from dKO mice were transduced to express wild-type (WT) TULA-2, the hyper-proliferative effect of dKO was dramatically reduced. This proliferation-suppressing effect of TULA-2 was reduced by various mutations within its phosphatase domain as well as within its UBA and SH3 domains, albeit the effects of UBA and SH3 mutations did not seem to be as profound 15. The effect of TULA-2 on Tcell proliferation correlated with its effect on TCR-induced tyrosine phosphorylation of

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Zap-70 and other proteins. Thus, transfection of TULA-2 in which its PTP activity was abolished by mutations into T cells exerted an opposite effect; in this case, Zap-70 and

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other TCR-induced phosphoproteins were phosphorylated to a higher degree than in WT cells 15. A similar effect was also shown for co-expression of Syk, a Zap-70 homologous

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PTK, and TULA-2 in HEK293T cells; co-expression of WT TULA-2 significantly reduced

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tyrosine phosphorylation of Syk, while co-expression of the mutant TULA-2 lacking PTP activity facilitated it 4,15,16.

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In contrast to the definitive results obtained with TULA-2, the role of PTP activity in the effects of TULA, the other TULA-family member, is less clear. Direct comparison

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of TCR-induced protein tyrosine phosphorylation in T cells from WT, TULA sKO, TULA-2 sKO and dKO mice confirmed that the effect of dKO substantially exceeds that of either sKO 19. However, a considerable difference between the two sKOs was observed. The effect of TULA-2 KO on tyrosine phosphorylation of Zap-70 and PLC was weaker than

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that of dKO, but still noticeable, whereas TULA sKO did not produce any detectable effect on phosphorylation of these proteins. TULA sKO and TULA-2 sKO exerted comparable effect only on the phosphorylation of Erk 19, a downstream element of T-cell signaling, which becomes activated through both Zap-70-dependent and –independent pathways and acts as a signal integrator 28-30. A similar difference was also observed for the biological effects of TULA sKO and TULA-2 sKO; the latter facilitated T-cell activation as judged by IFN- secretion, albeit weaker than did dKO, while the former

exerted no discernable effect 19. Consistent with this finding, overexpression of WT TULA-2 suppressed TCR-induced NF-AT activation in a T-cell line 31.

DIFFERENTIAL PTP ACTIVITY OF TULA AND TULA-2 The observed contrast between TULA and TULA-2 with regard to their effects on PTK-mediated signaling corresponds well to the differences in their enzymatic activity.

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TULA-2 has been shown to dephosphorylate phosphotyrosine (pY), but not phosphoserine or phosphothreonine in peptides and proteins 15,16. Furthermore, TULA-2

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dephosphorylates some phosphorylated small molecules, demonstrating a certain

degree of specificity; for example, it actively hydrolyzes p-nitrophenyl phosphate (pNPP)

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or fluorescein diphosphate (FDP), but not phenyl phosphate, inosine phosphates or

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phosphoglycerate 15,17,19. Specific activities of TULA and TULA-2 toward all these substrates differ greatly; pNPP is hydrolyzed by TULA-2 three to four orders of

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magnitude faster than by TULA 15,17. Likewise, pY-containing proteins are dephosphorylated by TULA and TULA-2 at drastically different rates. At physiological

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pH, TULA shows no discernable activity toward either total pY-containing proteins from TCR-stimulated T cells 17 or phosphorylated Syk 16, whereas TULA-2 dephosphorylates these substrates almost completely under these conditions. It has been shown that the optimal activity of TULA is seen at pH5 vs pH7 for TULA-2 and that at pH5 TULA

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dephosphorylates some protein bands among the total pY-containing proteins, but even at pH5 the specific activity of TULA appears to be at least two orders of magnitude lower than that of TULA-2 for these selected protein bands 17. Likewise, TULA activity toward pNPP remains four orders of magnitude lower that of TULA-2 even at pH5 17. The difference between specific activities of TULA and TULA-2 is less profound for some substrates, such as FDP or 3-O-methylfluorescein phosphate (OMFP), but even for them it is at least two orders of magnitude 19. In full agreement with a dramatic difference

between the enzymatic activities of TULA and TULA-2, multiple pY-containing peptide substrates have been readily identified for TULA-2, while no dephosphorylation of pYpeptides has been detected using the pY-peptide library screening for TULA 32. The observed difference appears to explain the opposite effects of these proteins upon overexpression of Syk with TULA and/or TULA-2 in HEK293T cells. TULA-2 reduced Syk-dependent phosphorylation as compared to that in the cells with

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endogenous TULA-2 only, while TULA increased it. Furthermore, TULA and TULA-2 were shown to compete in their effects on Syk upon their co-expression 16. Overall, the

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dominant-negative effect of TULA on Syk phosphorylation in the HEK293T

overexpression system resembled that of the PTP-inactivated [H380A]TULA-2; this

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observation was consistent with the lack of detectable in-vitro Syk-dephosphorylating

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activity for both [H380A]TULA-2 and WT TULA 16. This finding was, in its turn, consistent with the lack of Zap-70-dephosphorylating activity in vitro by [H380C]TULA-2 or WT

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TULA 19, and, in general, with the difference between specific activities of WT TULA-2, [H380A]TULA-2 and WT TULA toward pNPP; WT TULA-2 vs [H380A]TULA-2 differ by

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~9x104-fold, while WT TULA-2 vs WT TULA differ ~(2 to 6)x103 15,19. The phosphatase domains, including their catalytic pockets, of the two family

members are remarkably similar, and the key catalytic residues in both proteins are conserved and adopt conformations suitable for a phosphatase reaction

17.

It was

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speculated that the differences in activity between TULA and TULA-2 are due to certain non-catalytic non-conserved amino acid residues, and the mutational analysis supported the idea that making the active site of TULA more TULA-2-like improves the ability of this enzyme to hydrolyze pNPP, OMFP and pY-containing proteins 17,19. A recent study of human family members also showed a much higher specific activity of TULA-2 as compared to TULA, although this difference is less profound than between the mouse proteins 33. Key active-site residues are conserved between mouse and human proteins,

and even the sets of the non-conserved non-catalytic residues that are thought to underlie differences between mouse TULA and TULA-2 are mostly maintained in their human counterparts 17,33. However, some differences in non-catalytic residues between mouse and human TULA (~20% of their phosphatase domain sequences do differ) might explain the finding that the PTP activities of human TULA and TULA-2 differ less

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than those of the mouse family members.

OVERLAPPING YET DIFFERENTIAL EFFECTS OF THE TWO FAMILY MEMBERS

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The observed dramatic difference in the enzymatic activity between TULA and TULA-2 may explain the disparate effects of their sKOs discussed previously; only

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TULA-2 sKO, but not TULA sKO, causes a noticeable increase in TCR-induced Zap-70-

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mediated signaling 19. Hence, the notion of mostly overlapping effects of TULA and TULA-2 hinges on the finding that TULA/TULA-2 dKO exerts a substantially stronger

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effect on receptor signaling and cell activation than does TULA-2 sKO. However, the effect of TULA may be functionally complementary to that of TULA-2, but distinct by

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molecular mechanism. Some data suggest that the effect of TULA may be PTPindependent. Thus, TULA has been demonstrated to inhibit endocytosis apparently through dynamin sequestration 14. Also, binding of TULA to the apoptosis-inducing factor (AIF), a protein normally functioning in mitochondria as a FAD-dependent NADH

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oxidase, but exiting the mitochondria under stress conditions to trigger caspaseindependent cell death, and a TULA-dependent increase in T-cell death have been shown 34. However, it cannot be ruled out that a PTP activity is still critical for the regulatory effect of TULA, which may act here by dephosphorylating some yet-to-beidentified substrates. Our further understanding of this issue depends on the identification of substrates for TULA and TULA-2, an area also demonstrating a drastic disparity between TULA and TULA-2. Until now, the only protein substrates shown to be

dephosphorylated by TULA, albeit very slowly, are the two pY-containing unidentified bands of ca. 100 and 150kDa 17. In contrast, much is known about the substrate specificity of TULA-2, which is discussed in detail below. The differential effects of TULA and TULA-2 at the organismal level are even more complex than they are at the cellular level. As indicated above, rendering mouse T cells hyper-reactive to TCR ligation requires knocking out both family members 3,15. With

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regard to TCR signaling, T cells only from TULA-2 sKO, but not TULA sKO, exhibit a detectable difference as compared to WT T cells, although TULA-2 sKO does not

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facilitate signaling to the level characteristic for dKO 19. Hence, one may conclude that

the biological effect of either sKO should be much weaker than that of dKO and that the

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effect of TULA-2 sKO, if observed, is anticipated to be stronger than that of TULA sKO.

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However, comparison of WT and various TULA-family KOs in an experimental colitis model indicated that while dKO expectedly exhibited the strongest effect overall, both

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TULA sKO and TULA-2 sKO exerted detectable biological effects in which some clinical and laboratory parameters were different, whereas others, such as the inflammatory

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tissue damage, were very similar for the two sKOs and dKO 26. Moreover, multiple studies conducted in the last decade clearly demonstrated

association of certain single nucleotide polymorphisms (SNPs) in the genes encoding TULA (ubash3a) or TULA-2 (ubash3b) with several human diseases characterized by

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autoimmune and/or chronic inflammatory responses. The initial studies of the link between TULA- and TULA-2-encoding genes and human diseases were reviewed previously 8,9. More recent studies confirmed the initial observations and furthered molecular and genetic analysis of these phenomena

35,36.

In general, these diseases are

thought to be driven by T-cell responses, so defects in the ubash3b gene encoding TULA-2 might be expected to exert a more deleterious effect on human health. However, the absolute majority of the autoimmune/chromic inflammatory conditions

associated with defects in TULA-family genes are linked to the mutations in ubash3a; the only condition that appears to be dependent on some SNPs in ubash3b is Behçet's disease 37,38. Considering that TULA is primarily expressed in T cells (see above) and that the effect of TULA sKO in experimental has been fully blocked by the depletion of CD4+ T cells 26, the genetic results are in agreement with the notion of T-cell responses playing a key role in these conditions. On the other hand, a much less profound effect of

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the genetic variations in ubash3b, which encodes for TULA-2, is puzzling considering the abundance of findings made at the cellular level indicating that the effect of TULA-2

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SUBSTRATES OF TULA-FAMILY PTPs

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exceeds that of TULA.

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The initial studies indicated that TULA-2 dephosphorylates in vitro various pYcontaining proteins, including several PTKs: Src, Zap-70 15, EGFR, Cbl 39 and Syk 16.

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Consistent with these findings, TULA-2 reduced tyrosine phosphorylation of these PTKs as well as phosphorylation mediated by these PTKs in the cell 15,16,39. Subsequent

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studies employing screening of pY-peptide libraries revealed the substrate specificity of TULA-2 based on the nature of amino acid residues flanking the pY residues dephosphorylated by it; two classes of target motifs located N-terminally of the attacked pY were identified 32. The class I motif is defined by the presence of a proline residue at

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pY-1 and one or two aromatic residues mostly at the positions pY-3 and/or pY-4 as well as a dramatic reduction in the frequency of basic residues at pY-2 and also at pY-3. The class II motif has one or two aromatic residues mostly at pY-4 and/or pY-5 and one or two acidic ones at pY-2 and/or pY-3 (Figure 2); namely, 45% of the type II peptide substrates identified by screening of an N-terminal library had two aromatic and two acidic residues and 90% of these substrates had either one or two residues of each type. The exclusion of basic residues is even more severe in class II substrates than in

class I substrates; very few of the substrates identified contained a positively charged residue anywhere in the analyzed sequence. The C-terminal region of the pY-peptide substrates of TULA-2 also exhibits a significant increase in the frequency of aromatic and acidic residues and a strong exclusion of basic ones; 77% of the substrates identified by screening a C-terminal library feature one or two acidic and one or two aromatic residues within the region analyzed 32.

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The screening results were fully validated using subsequent kinetic studies with synthetic pY-peptides as substrates 32,40. Furthermore, in-vitro TULA-2 PTP assays

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using Syk, which contains multiple pY sites 41, as a substrate argued that the TULA-2

specificity determinants identified in pY-peptides remain to define TULA-2 specificity in

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full-length proteins 32. These studies indicated that Syk pY346, a regulatory site of this

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PTK 40,42-46, demonstrates the highest catalytic efficiency kcat/Km among the potential TULA-2 substrates examined that were derived from known protein sequences, while

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Syk pY519/pY520, a site considered to be a Syk activation marker 47-49 (numbering for murine sequences is used throughout the text), is a very poor substrate 32.

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The level of Zap-70 pY318, which is highly homologous to Syk pY346 and, likewise, a positive regulatory site 50-53, is also increased in cells in the absence of TULA2 19,26,54, and a synthetic peptide corresponding to the sequence containing Zap-70 pY318 and flanking residues is dephosphorylated by TULA-2 in vitro 31. All these results,

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taken together, supported the notion that Syk pY346/Zap-70 pY318 is a major physiological substrate site of TULA-2 and stimulated studies of the effect of TULA-2 on the regulation of Syk-family PTKs, which will be discussed in the last section. As indicated above some pY-containing proteins other than Syk and Zap-70 were

also shown to be substrates of TULA-2, including Src, EGFR and Cbl, although pY-sites of these substrates targeted by TULA-2 remain unidentified. These findings were supported by several studies in which modulation of pY content of these proteins was

demonstrated in the cell, although their dephosphorylation in vitro had not been shown. Thus, it was concluded, based on a study in B cells, that TULA-2 suppresses IFN-induced PI3K-mTOR signaling by dephosphorylating Syk and promotes IFN--induced JAK1-STAT1-mediated signaling by dephosphorylating Cbl 55. Similarly, the reason of an observed increase in the EGFR-dependent signaling in triple-negative breast cancer cells linked to the overexpression of TULA-2 was concluded to be a decrease in Cbl

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phosphorylation by TULA-2 56. A likely complex and/or indirect nature of the observed suppression of Cbl phosphorylation is demonstrated well by a study conducted in AML1-

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ETO cells, where RNAi-depletion of TULA-2 increased, consistent with the ability of

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TULA-2 to dephosphorylate Cbl, basal tyrosine phsphorylation of Cbl, but unexplectedly decreased Cbl phosphorylation upon treatment of these cells with a stimulatory mixture

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of cytokines 57. Another group of possible substrates of TULA-2 is represented by receptor PTKs c-Kit and Fms-like tyrosine kinase (FLT3). Co-expression of TULA-2, but

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not TULA, with these PTKs reduced tyrosine phosphorylation of both c-Kit and FLT3 in HEK293T cells in a PTP activity-dependent fashion, while tyrosine phosphorylation of

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FLT3 in FLT3-expressing 32D cells was increased by RNAi-depletion of TULA-2 58. Another oncogenic PTK dephophorylated by TULA-2 is Bcr-Abl. First, it was

reported previously that TULA-2 is part of the “core complex” around this PTK 59. It was recently shown that the level of Bcr-Abl p190 phosphorylation on Y226 and Y393

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(numbering for mouse Abl), markers of Bcr-Abl kinase activation, is decreased in cells transformed by Bcr-Abl in the presence of WT, but not PTP-inactivated TULA-2 60. In contrast, the general proteomics analysis of the effect of TULA-2 on the Bcr-Abl phosphoproteome and interactome indicated that the presence of TULA-2 moderately increased phosphorylation of Bcr-Abl p210 on the site corresponding to Abl Y393 61. In this study, the major Bcr-Abl pY-site negatively regulated by TULA-2 corresponded to

Bcr pY58 61. It should be noted that studies carried out in the cell, while providing valuable information about the phosphorylation status of multiple proteins in the physiologic context, do not identify specific substrates of PTPs. Thus, multiple pY-sites, especially those of Bcr-Abl-interacting proteins, were affected by RNAi-depletion of TULA-2, but this effect is likely to be complex and not entirely restricted to Bcr-Abl, because the TULA-2 knockdown greatly increases the level of Syk pY346 61, which up-

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regulates the kinase activity of Syk 40. The complexity of the effect of TULA-2 in this system was supported by the finding that phosphorylation of many pY-sites is not

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increased, but decreased following TULA-2 knockdown 61. The molecular mechanisms underlying the effect of TULA-2 on phosphorylation of Bcr-Abl and proteins interacting

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with it warrant further studies, because TULA-2 suppresses proliferation of Bcr-Abl-

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transplanted with such cells 60.

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transformed cells, while TULA/TULA-2 dKO reduces survival of experimental animals

UBA AND SH3 DOMAINS OF TULA PROTEINS: POSSIBLE ROLE IN SUBSTRATE

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BINDING

The TULA-2 substrate spectrum in the physiological context may be affected by

the presence of UBA and SH3 domains in the structure of TULA-family proteins. The SH3 domain mediates binding of TULA-family proteins to proline-rich motifs-containing SH3-binding proteins 4,5,14, while the UBA domain binds to ubiquitin and ubiquitylated

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proteins, including ubiquitylated TULA-family proteins themselves 4,5,11,12. One of the proteins binding to the TULA/TULA-2 SH3 domain is Cbl 4,5,14; this binding may be critical in rendering Cbl a substrate of TULA-2, because none of the major pY-sites of Cbl, i.e. pY698, pY737 or pY780 corresponding to human pY700, pY731 and pY774 possibly with an exception of pY737 (pY731), matches the consensus of a TULA-2 substrate site 32.

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Furthermore, it has been shown that TULA/TULA-2 dKO substantially increases the amount of proteins simultaneously ubiquitylated and tyrosine phosphorylated in response to TCR-mediated stimulation 13. A possible link between ubiquitylation of Zap70 and its dephosphorylation by TULA/TULA-2 has also been suggested in a study focused on the role of Nrdp1, an E3 ubiquitin-protein ligase, in T-cell activation. It has been concluded that Nrdp1 inhibits Zap-70 by ubiquitylating it, thus making it a substrate

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for TULA/TULA-2 63. Another case of the TCR-induced binding of TULA and TULA‐ 2 to the stimulation-dependent ubiquitin chains on Zap‐ 70 followed by dephosphorylation of

64.

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Zap‐ 70 has been reported in a study of the regulatory role of the Otud7b deubiquitinase These findings are consistent with the hypothesis that TULA-2 and, possibly, TULA

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bind to ubiquitylated pY-containing proteins and dephosphorylate them.

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Thus, the UBA and SH3 domains might be involved in the PTP activity of TULA-2 or both TULA and TULA-2 by bringing proteins that would otherwise be poorly

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recognized by TULA proteins to the proximity of their active site. Clearly, this role of UBA and SH3 in substrate recognition is warranted to be investigated further. However,

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several lines of evidence argue that the UBA and/or SH3 domains are unlikely to be critical for the functional interactions between TULA-2 and its substrates in general. First, binding of TULA and TULA-2 to Syk and TULA-2-dependent diminishing of Syk phosphorylation in the cell does not require the ubiquitylation of Syk, and nonubiquitylated Syk is dephosphorylated by TULA-2 in vitro 16. Likewise, TULA-2

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suppresses tyrosine phosphorylation of non-ubiquitylated Zap-70 in T cells 15,19. Second, the ability of TULA-2 to bind to Abl, the tyrosine phosphorylation of which is reduced by TULA-2 in the cell, does not require the presence of an intact UBA, SH3 or phosphatase domain in TULA-2 60. Third, TULA-2 effectively dephosphorylates multiple small pYcontaining peptide substrates lacking either SH3- or UBA-interacting sites and the sequences corresponding to these peptides in full-length proteins 32,40. Furthermore,

many studies of TULA-family proteins conducted using their recombinantly produced phosphatase domains indicate that the enzymatic properties of these domains and fulllength protiens are reasonably similar. For example, the drastic difference in specific activity between TULA and TULA-2 is observed for both isolated domains and full-length enzymes 15,19, while direct comparison indicates that the enzyme kinetics of the isolated TULA-2 PTP domain is similar, albeit not identical, to that of the full-length TULA-2 33.

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Also, both recombinant full-length TULA-2 and its recombinant PTP domain exhibit the ability to actively dephosphorylate Syk 16,21,32. Finally, the specificity toward individual pY-

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sites exhibited by TULA-2 in the cell closely resembles that revealed in the experiments with its isolated PTP domain 32,40.

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Taken together, these findings raise a question of the molecular mechanisms by

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which TULA-2 interacts with its substrates when this interaction is UBA- and SH3independent. Short pY-peptides are likely to interact directly with the PTP domain. This

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interaction depends on the amino acid residues flanking a pY being dephosphorylated; the flanking residues appear to interact with the active site and/or residues proximal to it,

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thus rendering dephosphorylation sequence-specific 32,40. Indeed, a substrate-trapping form of TULA-2 PTP lacking either UBA or SH3 binds well to both Zap-70 65 and Syk 40. However, full-length Syk binds to TULA and TULA-2 in the absence of cell stimulation and even in the absence of Syk detectable tyrosine phosphorylation 16, suggesting that

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TULA-family proteins and Syk may interact independent of the presence of pY in a substrate. Likewise, the interaction between TULA-2 and Bcr-Abl is independent of BcrAbl activation 60. Interactions of TULA-2 and Syk may be mediated by an adaptor protein as it was

shown for the stimulation of RBL-2H3 basophilic cells via FcRI, when the pY624/pY625 site of activated Syk becomes directly bound to Nck1, an adaptor protein, and SHIP-1, a lipid phosphatase. Then, formation of a large complex involving several proteins

including TULA-2 follows the initial binding of Nck1 and SHIP1 to Syk pY624/pY625

23.

However, considering the results discussed above, the pY-dependent Nck/SHIP-1mediated binding of TULA-2 to Syk likely represents recruitment of TULA-2 to the entire activation complex formed on the receptor for the subsequent regulation rather then the enzyme-substrate interaction between TULA-2 and Syk. The similar conclusion may be drawn from a recent study demonstrating binding

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of TULA to CIN85, an adaptor protein exerting a down-regulatory effect on TCR signaling and T-cell activation 54. CIN85 recruits TULA and Cbl to TCR in a stimulation-

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dependent manner, and the CIN85-TULA binding requires the intact CIN85 SH3 and proline-rich domains. The latter are likely to bind to the TULA SH3 domain, while the

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essential role of the CIN85 SH3 domain may be interpreted as the involvement of

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putative SH3-binding motifs on TULA. Alternatively, the CIN85 SH3 domain binds to Cbl, which, in turn, recruits TULA 54. It should be noted that the molecular mechanism of the

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down-regulatory effect of TULA on TCR signaling was not revealed in this study.

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REGULATION OF THE ACTIVITY OF TULA-FAMILY PTPs The UBA and SH3 domains, which are capable of various protein-protein

interactions, may potentially be involved not only in substrate recognition, but also in the regulation of TULA and/or TULA-2 activity, about which very little is currently known. It is

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clear that TULA-family proteins have basal phosphatase activity, which does not require either the presence of the UBA-SH3 region or any post-translational modifications, because their phosphatase domains obtained using bacterial expression exhibit high enzymatic activity 15-17,19,32, which for the isolated TULA-2 PTP was compared to that of its full-length form and found reasonably similar 33. Furthermore, TULA-2 immunoprecipitated from non-stimulated cells exhibited a high PTP activity 15,19.

Two potentially regulatory post-translational modifications of TULA-family proteins have been shown. First, phosphorylation was seen on TULA-2 Y8 (corresponding to Y19 in the human sequence) in response to TCR ligation 66. The role of this pY-site in the functions of TULA-2 remains unclear, although its proximity to the UBA domain suggests that it may affect protein-protein interactions mediated by this domain. Phosphorylation of TULA-2 was also reported in cells transformed with Bcr-Abl.

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Importantly, PTP-inactivated TULA-2 was phosphorylated much higher than was WT TULA-2, indicating that TULA-2 is capable of dephosphorylating itself 60.

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Second, ubiquitylation of TULA-family proteins has been shown 11,12. Mono-

ubiquitylation of TULA and TULA-2 results in the intramolecular binding of their UBA to

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the ubiquitin moiety on the same protein molecule, thus rendering the UBA domain

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incapable of binding to ubiquitin moieties on other proteins 11. Although the effect of this interaction on the PTP activity of TULA-family proteins has not been reported, it may

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substantially affect potential adaptor functions of TULA-family proteins. Regulation of enzymatic activity may also be mediated at the expression level,

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and such regulation has been shown for TULA-2 in multiple systems. In acute myeloid leukemia (AML) cell lines both transcriptional and microRNA (miR) control of TULA-2 was reported; miR-9-5p targeted TULA-2 in these cells 57. The miR-mediated regulation was also shown in triple-negative breast cancer cells, in which miR-200a targeted TULA2 56, and in megakaryocytes and megakaryocyte-like erythroleukemia cells where mIR-

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148a was shown to target TULA-2 67. Some recent studies were focused on the search for small-molecule inhibitors of

TULA-2 to be used as functional probes or as a basis for further drug development in order to manipulate biological responses dependent on this PTP. The phenylhydrazonopyrazolone sulfonate PHPS1, a general PTP inhibitor, has been shown

to significantly inhibit TULA-2 33. Subsequently, several inhibitors of this PTP that are both potent and selective have been identified using a high-throughput approach. 68

POSSIBLE ADAPTOR FUNCTIONS OF TULA-FAMILY PROTEINS Although adaptor functions of TULA-family proteins had not been studied extensively, the presence of interactive domains in their structure and binding of these

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domains to many biologically important proteins suggest that TULA-family proteins may also function as adaptors. This notion is supported by several studies pointing out some

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functions of TULA and TULA-2 that appear to be independent of their PTP activity. For

instance, TULA binds to the AIF protein and facilitates the AIF-dependent cell death 34.

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Binding to AIF and up-regulation of AIF-dependent cell death is specific for TULA, which

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has a much lower PTP activity than TULA-2, and the N-terminal half of TULA lacking the phosphatase domain binds to AIF better than full-length TULA, suggesting that the

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interaction of TULA with AIF is independent of its PTP activity 34. Furthermore, neither UBA nor SH3 of TULA is essential for its binding to AIF, whereas the biological effect of

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TULA in this system is dependent on both UBA and SH3, thus suggesting that the binding of TULA to yet-unidentified proteins is essential for its positive effect on cell death.

The results of a recent study demonstrating a role of TULA in rheumatoid arthritis

development appear to be consistent with the finding linking TULA to AIF and cell death.

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The number of IL-2-producing CD4+ T cells is increased in the spleen of TULA sKO mice, which show a higher incidence of collagen type II-induced autoimmune arthritis as compared to WT mice. Notably, proliferation of CD4+ T cells remains unaffected in TULA sKO mice, whereas their resistance to growth factor withdrawal-induced cell death is modestly, but significantly increased 27.

Another biological function of TULA-family apparently independent of their PTP activity is related to the ability of these proteins to bind to ABCE-1, a cellular protein factor of HIV-1 assembly, and suppress the late stages of HIV-1 production. The UBA domain, while not contributing to the TULA-ABCE-1 binding, is essential for this effect of TULA, suggesting that TULA-family proteins inhibit HIV-1 biogenesis by interfering with a UBA-ubiquitin interaction critical for this process. This finding together with the finding

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that both TULA and TULA-2 inhibit HIV-1 biogenesis to a similar degree in spite of the

HIV-1 biogenesis through adaptor-type interactions 69.

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profound differences in their PTP activities argue that the TULA-family proteins modulate

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The down-regulatory effect of TULA on the TCR-induced NF-B pathway, which manifests itself by a TULA sKO-mediated increase in the activity of IKK kinase complex

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and IKK activity-dependent degradation of I-B, appears to depend on the SH3 and UBA domains of TULA. The SH3 domain of TULA binds to TAK1 and NEMO, key

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components of the system of IKK activation, and UBA binds to Lys63- and Met1-type ubiquitin chains, which are critical for this activation 12. However, the involvement of the

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N-terminal domains of TULA in these effects does not rule the involvement of its PTP activity, since this issue has not been examined directly in this study. TULA-2 has been shown to form a complex with the mitotic kinesin-like protein 2

(MKlp2) and, in cooperation with the latter, to recruit Aurora B, a protein kinase

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regulating chromosomal segregation, from the centromeres to the spindle microtubules, thus inducing anaphase 70. The interaction with Aurora B, which is ubiquitylated, is mediated by the UBA domain of TULA-2, although the structural basis of TULA-2 binding to MKlp2 remains unknown. The role of PTP activity in this process has not been studied, but it is possible that TULA-2 functions in it as an adaptor protein. Interestingly, TULA-family proteins also bind to Cbl 4,5, and Cbl has been shown to exert an effect on

microtubules 71. However, the involvement of Cbl in the effect of TULA-2 on chromosomal segregation has not been investigated. TULA-2 was also shown to bind to the adaptor protein ShcA 72. Specific binding of TULA-2 to a peptide containing the ShcA pY317 site and an increase in TULA-2-ShcA binding following the EGFR-mediated tyrosine phosphorylation of ShcA suggest that the interaction between TULA-2 and ShcA is mediated by binding of a pY-site to the

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phosphatase domain of TULA-2 72. However the interaction of TULA-2 to ShcA pY317 has not been shown directly, and it remains unclear why TULA-2 does not

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dephosphorylate this site, since the flanking regions of ShcA pY317 are consistent with it

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being a reasonably good substrate for TULA-2 32.

REGULATORY EFFECT OF TULA-2

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REGULATION OF SYK-FAMILY PTKs: THE BEST-CHARACTERIZED

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Based on the findings outlined above one may conclude that regulation of Syk and Zap-70 in the cells where they play a key role in the receptor signal transduction is,

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to-date, the best-characterized effect of TULA-2 and, in general, of TULA-family proteins, on signaling. The data on TULA-2 substrate specificity strongly argue that Syk pY346 is the best known substrate site of TULA-2 on Syk 32 (Fig. 3). Since pY346 is one of the key regulatory sites of Syk 42-46, its role in the down-regulation of Syk and Sykmediated signaling by TULA-2 was evaluated in platelets and in a reconstituted system

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mimicking platelet signaling via their GPVI receptor for collagen 40. For GPVI signaling reconstitution, HEK293T cells were co-expressing a chimeric receptor containing the cytoplasmic tail of FcR, a signal-transducing part of the GPVI complex, which contains a immunoreceptor tyrosine-based activation motif (ITAM), with TULA-2 and Syk – either WT or mutant lacking some key regulatory sites in various combinations 40. The study was focused on platelets, because the protein level of TULA-2 in these cells is extremely

high, whereas TULA is not detectable by immunoblotting, and because the lack of TULA-2 was previously shown to greatly facilitate GPVI/FcR-depenent signaling as well as the resulting physiological responses of platelets in vitro and in vivo, including aggregation, secretion and thrombus formation 21,32. Only sKO of TULA-2, but not that of undetectable TULA, enhances Syk activation, Syk-mediated signaling and biological responses in platelets 40. Further

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results argue that TULA-2 controls Syk activation in response to GPVI ligation by actively dephosphorylating Syk pY346, which is formed upon platelet stimulation faster

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than Syk pY342 or Syk pY519/pY520, the other pY-sites examined in this study 40.

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Previous reports indicated that phosphorylation of Syk Y346 in mast cells is mostly independent of Syk enzymatic activity, but mediated instead by Lyn, a Src-family PTK,

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whereas Syk Y342 is mostly autophosphorylated 73.

The down-regulation of Syk by TULA-2 was effective only for the moderate

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degree of stimulation, while strong stimulation was not significantly suppressed 21,40. It appears that phosphorylation of Syk Y342, the site that neighbors pY346 and also plays

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a positive role in the activation of Syk 40,43,44,74, but is dephosphorylated by TULA-2 much less efficiently than pY346, allowed Syk to progress along its activation sequence as shown in the reconstitution system using mutational analysis. Furthermore, phosphorylation of Syk Y342 renders the existing Syk pY346 a substantially weaker

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substrate of TULA-2 than Syk pY346 is in the absence of pY342. Thus, phosphorylation of Syk Y342 may be key for overcoming the down-regulation of Syk by TULA-2 through both directly promoting Syk activation and protecting pY346 from dephosphorylation by TULA-2 40. The model based on these findings is presented in Fig. 3. Notably, the overall strong down-regulatory effect of TULA-2 on GPVI-mediated signaling and biological responses is caused mostly by the highly specific effect of TULA-2 on Syk activation, which is essential for triggering the entire pathway, since Syk

is the only protein among the major platelet signaling proteins examined that detectably binds to a substrate-trapping form of TULA-2. Furthermore, the absolute majority of pYcontaining protein material bound to substrate-trapping TULA-2 co-migrates with Syk 40. Although these results do not rule out the existence of other key substrates of TULA-2, they sustain the notion that inhibition of Syk, a highly specific effect of TULA-2, may result in down-regulation of the entire signaling cascade.

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The molecular mechanisms of the down-regulatory effect of TULA-2 has been studied for the GPVI/FcR complex in more detail than for other receptors mediating

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signaling in platelets, but this effect of TULA-2 is not specific for GPVI/FcRIt has been

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shown that phosphorylation of Syk in response to the ligation of FcRIIA, an ITAMcontaining receptor for IgG, is enhanced by RNAi-depletion of TULA-2 in

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erythroleukemia cells, while reduced by anti-miR-148a, which increases the level of TULA-2 in these cells. Furthermore, anti-miR-148a reduced calcium mobilization and

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integrin activation in this system, and rendered mice more resistant to FcRIIA-mediated thrombosis 67. Consistent with these findnings, a decrease in the level of TULA-2 in

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heterozygous WT/sKO mice facilitated Syk phosphorylation, Syk-dependent signaling, integrin activation and aggregation of platelets in reponse to FcRIIA ligation as compared to those in platelets from WT mice. A decrease in the level of TULA-2 also reduces the tail bleeding time and exacerbates the heparin-induced thrombocytopenia

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(HIT)-like condition in mice 75.

The effect of TULA-2 on Syk-dependent signaling consistent with the systems in

which cells were stimulated through ITAM-containing receptors was also reported in platelets stimulated through the C-type lectin-like receptor 2 (CLEC-2), which bears a motif similar to one half of an ITAM, termed a hem-ITAM. Dimerization of hem-ITAM receptors is likely to juxtapose two hem-ITAMs arranging them in an ITAM-resembling

structure 76,77. The lack of TULA-2 increased Syk phosphorylation, including its phosphorylation on Syk Y346, as well as Syk-dependent signaling and biological functions of platelets in response to CLEC-2 ligation 78. This and other findings in the studies of platelet signaling indicated that TULA-2 strongly down-regulates various pathways involving Syk as a key signal-transducing component 21,32,40,67,75,78, while not those largely independent of Syk, such as thrombin-induced responses 21,67. The

receptors that are suppressed by TULA-2 are presented in Table I.

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individual events of platelet signaling induced by various ITAM- or hemITAM-bearing

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Likewise, it has been established that Zap-70 plays a key role in TCR-triggered

signaling 79 and that TULA-2 dephoshorylates Zap-70 pY318, a regulatory site of Zap-70 homologous to Syk pY346 19,26. Together, these findings suggest that the effect of

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50-53

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TULA-2 on Zap-70-mediated signaling in T cells should be similar to that on Sykdependent signaling in platelets. However, they are clearly different, since TULA-2 sKO

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exerts an effect significantly lower than that of TULA/TULA-2 dKO 3,15,19, indicating that TULA is also essential for the suppression of TCR-induced Zap-70-dependent activation

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of T cells. As discused above, this may be related to the effect of TULA on regulatory events other than Zap-70-mediated signaling. The observed difference between the Zap70 regulation in T cells and the Syk regulation in platelets is not unexpected, since the regulatory mechansisms for Syk and Zap-70 differ in some aspects: Syk appears to be more dependent on autophosphorylation and less dependent on Src-family PTKs than

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Zap-70 45,80,81.

Notably, the inhibitory effect of TULA-2 on Syk regulates signaling not only in

platelets, but in all cells where TULA-2 is expressed and where Syk plays a key role in signaling. Thus, it has been shown that RNAi-depletion of TULA-2 down-regulates the FcRI receptor-induced activity of Syk as well as Syk-mediated tyrosine phosphorylation, transcription factor activation and degranulation in mast cells 23. Likewise, TULA-2

suppresses the ITAM-induced Syk activation and Syk-mediated tyrosine phosphorylation in bone marrow-derived macrophages and supresses functions of osteoclasts, boneresorbing cells of the macrophage lineage, in a PTP activity-dependent manner 25. It is interesting whether or not the observed effect of TULA-2 on the osteoclast function is in any way related to a recently reported association between SNP variants of ubash3b, a TULA-2-encoding gene, and the variations in formation of facial shape in individual

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development, a process likely linked to bone remodelling 82,83. Another important system, in which TULA-family proteins are critically involved in

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the receptor signaling and the resulting physiological responses, is anti-fungal immunity; dKO mice lacking both TULA and TULA-2 are significantly more resistant to the systemic

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infection by Candida albicans 22,84. The effect of dKO is largerly cell-autonomous, since it

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is reproduced by the transfer of dKO bone marrow cells to WT recipient mice 22. Moreover, bone marrow monocytes and bone marrow-derived DCs obtained from dKO

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mice show the higher anti-fungal activity in vitro than WT cells. This increase appears to be caused by the enhancement of the activitation of Syk through dectin-1, a major

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receptor for -glucan, which accounts for a substantial portion of the fungal cell wall, including that of C. albicans. The enhancement of Syk activity caused by dKO upregulates PLC2 phosphorylation and the production of reactive oxigen species 22. Notably, the lack of TULA alone also increases the resistance to C. albicans in vivo,

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although to a weaker extent than does dKO 84, in spite of TULA being mostly a lymphoid protein, which is not or poorly expressed in phagocytes (see “Structure and Expression of TULA proteins”). These results are consistent with the significant involvement of T-cell immune responses to the host defense against C. albicans 85-88.

CONCLUSIONS

The studies focused on the functions of TULA-family proteins have demonstrated that these proteins act as key regulators of cellular activation. The molecular mechanism of the regulatory effect is better understood for TULA-2, a family member exhibiting the higher PTP activity; dephosphorylation of a major regulatory pY-site on Syk and Zap-70 PTKs appears to be mostly responsible for the inhibitory effect of TULA-2 on cellular signaling. Dephosphorylation of Syk pY346, a site exhibiting the excellent subtrate

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properties for TULA-2, has been shown to suppress activation of Syk and to downregulate the entire Syk-mediated signalling pathway in platelets stimulated through ITAM

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or hemITAM-containing receptors. It is likely that various signaling pathways, in which Syk plays a key role, are also suppressed by TULA-2 in other cells. The extent of this

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effect may be dependent on the level of TULA-2 PTP activity relative to the levels of

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other PTPs dephosphorylating Syk and other signaling proteins; among various cell types characterized to date platelets show the highest level of TULA-2, but, in general,

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TULA-2 is expressed ubiquitously. Taken together with the critical involvement of Syk in multiple signaling pathway, the down-regulatory effect of TULA-2 may be of universal

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importance and, thus, play a key role in the development of many clinical conditions. Although at the molecular level the effect of TULA-2 on Zap-70 appears to be

similar to the effect of TULA-2 on Syk, the mechanisms mediating down-regulatory functions of TULA-family proteins in T cells are more complex, since TULA is an

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essential element of the suppressive effect of TULA-family proteins on T-cell activation. Similar to TULA-2, TULA exerts a negative effect on TCR-triggered signaling, but the molecular basis of its effect has not been fully deciphered yet. It cannot be ruled out that the down-regulatory effect of TULA is unrelated to its PTP activity. It is also possible that the effect of TULA is due to dephosphorylation of the yet-unidentified substrate(s). Thus, the current focus on the Syk-TULA-2 interactions resulting from the finding that Syk pY346 is extremely sensitive to dephosphorylation by TULA-2 may change when

additional biologically relevant substrates of TULA-2 are characterized. For instance, Bcr-Abl, an oncogenic PTK, may represent one of such substrates; although the molecular basis of its dephosphorylation is less clear, clinical importance of this regulatory event is potentially very high. Taken together, the studies conducted in the last decade or so clearly indicate that the TULA protein family plays a key regulatory role, which appears to be mostly

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mediated by their effects on cellular signaling. In addition to being critical for normal cellular functions, these regulatory effects appear to be involved in multiple clinical

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conditions, mostly related to autoimmunity and/or chronic inflammation. The molecular basis of these effects has not been fully determined, but down-regulation of multiple

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receptor-initiated signaling pathways, especially those mediated by Syk PTK, clearly

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plays a key role in the regulatory effects of TULA-2.

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ACKNOWLEDGEMENTS

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The author thanks Carol A. Dangelmaier for excellent editorial help.

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FIGURE LEGENDS

Figure 1. Structure of TULA‐ family proteins. Major functional domains of UBASH3/STS/TULA proteins are shown, including ubiquitin‐ associated domain (UBA), Src‐ homology domain 3 (SH3), histidine phosphatase domain and the dimerization sequence, using mouse UBASH3B/STS-

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1/TULA-2 as an example. Sequence similarity between TULA and TULA-2 inside the domains indicated is shown

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as percentage of identical plus positive amino acid residues. The key catalytic histidine

of the phosphatase domain is shown. The 2H domain whose function in the TULA-family

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proteins remains unknown is also shown. The conservative domain search tool

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recognizes this region as either the 2’-5’ RNA ligase or LigT, which are both related to

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2H superfamily 20, in some, but not all, TULA-2 and invertebrate TULA-like proteins.

Figure 2. Sequence specificity determinants for TULA-2 substrates. Class I N-terminal motif is defined by the presence of a proline residue at position pY-1. It requires also the presence of one or two aromatic residues and the exclusion of basic

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residues at certain positions. Preference (green) or aversion (red) of various types of amino acid residues at individual positions is denoted by the letter case: strong and weaker preference/aversion are indicated as uppercase and lowercase, respectively. Class II N-terminal motif shows a strong preference for aromatic and acidic residues at positions pY-5 and pY-4 (aromatic) and pY-3 and pY-2 (acidic). Positions pY-3 and pY-2 show also some preference for aromatic residues. Also, a very strong aversion of basic

residues for the entire length of the analyzed sequence is seen in class II. C-terminal substrate sequences show, likewise, a strong preference for aromatic and acidic residues and a strong aversion of basic ones, but no discernable motif is recognized in

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of

them.

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Figure 3. Effect of TULA-2 on Syk activation: Working model.

Non-phosphorylated Syk exists in the auto-inhibited conformation, which changes to

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active or partially active following binding of the Syk tandem SH2 domains to a phosphoITAM or phospho-hemITAMs. As a result of this change, the regulatory tyrosines of Syk

lP

become accessible for phosphorylation. Among the pY-sites depicted, Syk pY346 is formed first, mostly as a result of phosphorylation by Src-family PTKs, while TULA-2

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dephosphorylates it, inhibiting further activation of Syk. When pY342 is formed, mostly by auto-phosphorylation, the ability of TULA-2 to dephosphorylate pY346 diminishes, and both Y342 and Y346 become phosphorylated, thus stabilizing the active conformation. Finally, the pY519/pY520 site is formed, and Syk becomes fully activated.

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Only the initial binding of Syk to phospho-ITAM or phospho-hemITAM is shown for the sake of brevity.

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Table 1. Reported inhibition of early signaling events in platelets stimulated via the ITAM- or hemITAM-bearing receptors GPVI/FcR, FcRIIA and CLEC-2 by TULA-2 Reports

Phosphorylation of Syk

20,31,39,66,74,77

Phosphorylation of SLP-76

39,77

Phosphorylation of LAT

74

Phosphorylation of Cbl

39

Phosphorylation of Btk

31

Phosphorylation of PLC-2

20,39,74,77

Ca2+ mobilization

20,66,77

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Signaling event