Dara K. Mohammad

TEC family kinases in health and disease – loss‐of‐function of BTK and ITK and the gain‐of‐function fusions ITK–SYK and BTK–SYK

The TEC family is ancient and constitutes the second largest family of cytoplasmic tyrosine kinases. In 1993, loss‐of‐function mutations in the BTK gene were reported as the cause of X‐linked agammaglobulinemia. Of all the existing 90 tyrosine kinases in humans, Bruton’s tyrosine kinase (BTK) is the kinase for which most mutations have been identified. These experiments of nature collectively provide a form of mutation scanning with direct implications for the several hundred endogenous signaling proteins carrying domains also found in BTK. In 2009, an inactivating mutation in the ITK gene was shown to cause susceptibility to lethal Epstein–Barr virus infection. Both kinases represent interesting targets for inhibition: in the case of BTK, as an immunosuppressant, whereas there is evidence that the inhibition of inducible T‐cell kinase (ITK) could influence the infectivity of HIV and also have anti‐inflammatory activity. Since 2006, several patients carrying a fusion protein, originating from a translocation joining genes encoding the kinases ITK and spleen tyrosine kinase (SYK), have been shown to develop T‐cell lymphoma. We review these disease processes and also describe the role of the N‐terminal pleckstrin homology–Tec homology (PH–TH) domain doublet of BTK and ITK in the downstream intracellular signaling of such fusion proteins.

Dual Phosphorylation of Btk by Akt/Protein Kinase B Provides Docking for 14-3-3ζ, Regulates Shuttling, and Attenuates both Tonic and Induced Signaling in B Cells

ABSTRACT Brutons tyrosine kinase (Btk) is crucial for B-lymphocyte activation and development. Mutations in the Btk gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Using tandem mass spectrometry, 14-3-3ζ was identified as a new binding partner and negative regulator of Btk in both B-cell lines and primary B lymphocytes. The activated serine/threonine kinase Akt/protein kinase B (PKB) phosphorylated Btk on two sites prior to 14-3-3ζ binding. The interaction sites were mapped to phosphoserine pS51 in the pleckstrin homology domain and phosphothreonine pT495 in the kinase domain. The double-alanine, S51A/T495A, replacement mutant failed to bind 14-3-3ζ, while phosphomimetic aspartate substitutions, S51D/T495D, caused enhanced interaction. The phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY294002 abrogated S51/T495 phosphorylation and binding. A newly characterized 14-3-3 inhibitor, BV02, reduced binding, as did the Btk inhibitor PCI-32765 (ibrutinib). Interestingly, in the presence of BV02, phosphorylation of Btk, phospholipase Cγ2, and NF-κB increased strongly, suggesting that 14-3-3 also regulates B-cell receptor (BCR)-mediated tonic signaling. Furthermore, downregulation of 14-3-3ζ elevated nuclear translocation of Btk. The loss-of-function mutant S51A/T495A showed reduced tyrosine phosphorylation and ubiquitination. Conversely, the gain-of-function mutant S51D/T495D exhibited intense tyrosine phosphorylation, associated with Btk ubiquitination and degradation, likely contributing to the termination of BCR signaling. Collectively, this suggests that Btk could become an important new candidate for the general study of 14-3-3-mediated regulation.

Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54)

ABSTRACT Brutons tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btks SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner.

Anti-leukaemic effects induced by APR-246 are dependent on induction of oxidative stress and the NFE2L2/HMOX1 axis that can be targeted by PI3K and mTOR inhibitors in acute myeloid leukaemia cells

The small molecule APR‐246 (PRIMA‐1MET) is a novel drug that restores the activity of mutated and unfolded TP53 protein. However, the mechanisms of action and potential off‐target effects are not fully understood. Gene expression profiling in TP53 mutant KMB3 acute myeloid leukaemia (AML) cells showed that genes which protected cells from oxidative stress to be the most up‐regulated. APR‐246 exposure also induced reactive oxygen species (ROS) formation and depleted glutathione in AML cells. The genes most up‐regulated by APR‐246, confirmed by quantitative real time polymerase chain reaction, were heme oxygenase‐1 (HMOX1, also termed HO‐1), SLC7A11 and RIT1. Up‐regulation of HMOX1, a key regulator of cellular response to ROS, was independent of TP53 mutational status. NFE2L2 (also termed Nrf2), a master regulator of HMOX1 expression, showed transcriptional up‐regulation and nuclear translocation by APR‐246. Down‐regulation of NFE2L2 by siRNA in AML cells significantly increased the antitumoural effects of APR‐246. The PI3K inhibitor wortmannin and the mTOR inhibitor rapamycin inhibited APR‐246‐induced nuclear translocation of NFE2L2 and counteracted the protective cellular responses to APR‐246, resulting in synergistic cell killing together with APR‐246. In conclusion, ROS induction is important for antileukaemic activities of APR‐246 and inhibiting the protective response of the Nrf‐2/HMOX1 axis using PI3K inhibitors, enhances the antileukaemic effects.

Signaling of the ITK (Interleukin 2-inducible T Cell Kinase)-SYK (Spleen Tyrosine Kinase) Fusion Kinase Is Dependent on Adapter SLP-76 and on the Adapter Function of the Kinases SYK and ZAP70 *

Background: ITK-SYK is an oncogenic fusion protein in patients with peripheral T cell lymphomas carrying a unique translocation. Results: Mutations in critical tyrosines disable the constitutive activity of ITK-SYK. Intracellular signaling of ITK-SYK requires both SLP-76 and the adapter function of SYK/ZAP-70 kinases. Conclusion: The ITK-SYK fusion protein is dependent on adapters. Significance: This study provides insight into the activation and signaling of ITK-SYK. The inducible T cell kinase-spleen tyrosine kinase (ITK-SYK) oncogene consists of the Tec homology-pleckstrin homology domain of ITK and the kinase domain of SYK, and it is believed to be the cause of peripheral T cell lymphoma. We and others have recently demonstrated that this fusion protein is constitutively tyrosine-phosphorylated and is transforming both in vitro and in vivo. To gain a deeper insight into the molecular mechanism(s) underlying its activation and signaling, we mutated a total of eight tyrosines located in the SYK portion of the chimera into either phenylalanine or to the negatively charged glutamic acid. Although mutations in the interdomain-B region affected ITK-SYK kinase activity, they only modestly altered downstream signaling events. In contrast, mutations that were introduced in the kinase domain triggered severe impairment of downstream signaling. Moreover, we show here that SLP-76 is critical for ITK-SYK activation and is particularly required for the ITK-SYK-dependent phosphorylation of SYK activation loop tyrosines. In Jurkat cell lines, we demonstrate that expression of ITK-SYK fusion requires an intact SLP-76 function and significantly induces IL-2 secretion and CD69 expression. Furthermore, the SLP-76-mediated induction of IL-2 and CD69 could be further enhanced by SYK or ZAP-70, but it was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases. Altogether, our data suggest that ITK-SYK exists in the active conformation state and is therefore capable of signaling without SRC family kinases or stimulation of the T cell receptor.

ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

Bruton’s Tyrosine Kinase (BTK) is a cytoplasmic protein tyrosine kinase with a fundamental role in B-lymphocyte development and activation. The nucleocytoplasmic shuttling of BTK is specifically modulated by the Ankyrin Repeat Domain 54 (ANKRD54) protein and the interaction is known to be exclusively SH3-dependent. To identify the spectrum of the ANKRD54 SH3-interactome, we applied phage-display screening of a library containing all the 296 human SH3 domains. The BTK-SH3 domain was the prime interactor. Quantitative western blotting analysis demonstrated the accuracy of the screening procedure. Revealing the spectrum and specificity of ANKRD54-interactome is a critical step toward functional analysis in cells and tissues.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Translocation-generated ITK-FER and ITK-SYK fusions induce STAT3 phosphorylation and CD69 expression

Many cancer types carry mutations in protein tyrosine kinase (PTK) and such alterations frequently drive tumor progression. One category is gene translocation of PTKs yielding chimeric proteins with transforming capacity. In this study, we characterized the role of ITK-FER [Interleukin-2-inducible T-cell Kinase (ITK) gene fused with Feline Encephalitis Virus-Related kinase (FER) gene] and ITK-SYK [Interleukin-2-inducible T-cell Kinase (ITK) gene fused with the Spleen Tyrosine Kinase (SYK)] in Peripheral T Cell Lymphoma (PTCL) signaling. We observed an induction of tyrosine phosphorylation events in the presence of both ITK-FER and ITK-SYK. The downstream targets of ITK-FER and ITK-SYK were explored and STAT3 was found to be highly phosphorylated by these fusion kinases. In addition, the CD69 T-cell activation marker was significantly elevated. Apart from tyrosine kinase inhibitors acting directly on the fusions, we believe that drugs acting on downstream targets could serve as alternative cancer therapies for fusion PTKs.

Terminating B cell receptor signaling

Bruton’s tyrosine kinase (BTK) mediated signaling is critical for B cell development and activation. The physiological role of BTK is evident since its functional loss results in a differentiation block of B cells beyond the pro-B cell stage causing X-linked agammaglobulinemia. Inhibitors of BTK, such as ibrutinib and acalabrutinib profoundly impair BCR signaling. They are highly effective in the treatment of many lymphoid tumors, including chronic lymphocytic leukemia, mantle cell lymphoma, Waldenström’s macroglobulinemia and marginal zone lymphoma. Most patients respond, but escape mutants of different origins may appear [13]. The most common mutations affect BTK itself, impairing the binding of the inhibitors to cysteine 481 in the kinase domain. Others, instead, introduce changes in Phospholipase Cγ2 (PLCγ2), which is the major substrate of BTK, rendering this enzyme constitutively active and thereby signaling independently of BTK/BCR. In light of the potent effect of BTK inhibitors, it seems important to consider the endogenous/ physiological control of the BCR signaling pathway, including how activated BTK is turned off. Immediately upstream of BTK are SRC-family kinases (SFKs). They phosphorylate tyrosine 551 in BTK, which changes the conformation leading to an activated form of the kinase. In B lymphocytes LYN is the predominant member of this family (Figure 1, panel A), while also BLK and FYN are expressed. SFKs are regulated in a unique way through the phosphorylation of a conserved C-terminal tyrosine. Upon this phosphorylation by the CSK kinase, the modified tyrosine tethers to the tyrosine-binding SH2 domain in cis, thereby impeding enzyme activity. This phosphorylation is reversible and the CD45 transmembrane phosphatase is directly involved [4]. Many tyrosine kinases, and not only SFKs, are themselves regulated by various forms of reversible tyrosine phosphorylation mediated by other kinases and phosphatases. BTK regulation seems to differ, as up until today, there is no report demonstrating tyrosine dephosphorylation of BTK by phosphatases. Instead, it seems as if serine/threonine kinases (S/T kinases) are involved in the down-regulation of BCR signaling, often with 14-3-3 proteins being crucial contributors to this process. Examples of the BCR downstream proteins involved in this process are BTK, Spleen tyrosine kinase (SYK) and B cell linker/adapter protein (BLNK) [5]. Thus, we have previously shown that the S/T kinase, protein kinase B (AKT/PKB), phosphorylates two sites in BTK, one located in the Pleckstrin homology (PH) domain and the other in the kinase domain [5]. 14-33 proteins are small and acidic, lack enzymatic activity and naturally occur as dimers. Dual S/T phosphorylations make molecules ideal targets for this family of proteins. Binding of 14-3-3 proteins to BTK triggers proteasomal degradation (Figure 1). Hence, following activation by the generation of pY551, the activated BTK molecules are selectively subjected to robust S/T phosphorylation and subsequently targeted for degradation. In further support of the idea that S/T kinases downregulate BCR signaling is the fact that Protein kinase C (PKC) β abates the activity of BTK by phosphorylating serine 180 in the Tec homology (TH) domain [6]. The underlying mechanism seems to impair membrane translocation and the subsequent phosphorylation of pY551, which is required for activation of BTK. Editorial

Protein kinase B (AKT) regulates SYK activity and shuttling through 14-3-3 and importin 7

The Protein kinase B (AKT) regulates a plethora of intracellular signaling proteins to fine-tune signaling of multiple pathways. Here, we found that following B-cell receptor (BCR)-induced tyrosine phosphorylation of the cytoplasmic tyrosine kinase SYK and the adaptor BLNK, the AKT/PKB enzyme strongly induced BLNK (>100-fold) and SYK (>100-fold) serine/threonine phosphorylation (pS/pT). Increased phosphorylation promoted 14-3-3 binding to BLNK (37-fold) and SYK (2.5-fold) in a pS/pT-concentration dependent manner. We also demonstrated that the AKT inhibitor MK2206 reduced pS/pT of both BLNK (3-fold) and SYK (2.5-fold). Notably, the AKT phosphatase, PHLPP2 maintained the activating phosphorylation of BLNK at Y84 and increased protein stability (8.5-fold). In addition, 14-3-3 was required for the regulation SYKs interaction with BLNK and attenuated SYK binding to Importin 7 (5-fold), thereby perturbing shuttling to the nucleus. Moreover, 14-3-3 proteins also sustained tyrosine phosphorylation of SYK and BLNK. Furthermore, substitution of S295 or S297 for alanine abrogated SYKs binding to Importin 7. SYK with S295A or S297A replacements showed intense pY525/526 phosphorylation, and BLNK pY84 phosphorylation correlated with the SYK pY525/526 phosphorylation level. Conversely, the corresponding mutations to aspartic acid in SYK reduced pY525/526 phosphorylation. Collectively, these and previous results suggest that AKT and 14-3-3 proteins down-regulate the activity of several BCR-associated components, including BTK, BLNK and SYK and also inhibit SYKs interaction with Importin 7.