Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

Abstract

Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK–SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK–SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK–SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK–SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK–SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

Introduction

Spleen tyrosine kinase (SYK) is a non-receptor protein–tyrosine kinase [1] expressed in a wide variety of tissues, including epithelial and immune cells [2], [3]. It behaves as a tumor suppressor protein in breast and gastric cancers, while, in contrast, it renders B and T-cell lymphomas as well as head and neck cancers apoptosis resistant [4], [5], [6]. Chromosomal translocations are frequently oncogenic [7], and over-expressed, or deregulated SYK, causes tumors. Thus, SYK is known to fuse with the dimeriser protein TEL, forming a constitutively active chimera TEL–SYK [8]. Recently, the SYK gene was shown to be involved in another translocation event t(5;9)(q33;q22) with the ITK gene encoding the IL-2-dependent T-cell kinase, yielding a fusion-protein ITK–SYK in a subset of peripheral T-cell lymphomas [9], [10], [11]. ITK is a Tec family member transducing signals from multiple receptors, including the T-cell receptor TCR [12], [13], [14]. ITK has an amino-terminal PH (pleckstrin homology), a TH (Tec homology) domain, which contains both a Zn⁺² binding motif and a proline-rich region (PRR), followed by SH3, SH2, and a kinase domain [15], [16], [17].

To our knowledge, ITK–SYK is the only kinase-to-kinase, exon-to-exon, in-frame chimeric protein, which is the aftermath of a reciprocal translocation between two kinases [9]. The resulting fusion-protein has replaced the N-terminal tandem SH2 domains and a small part of linker B of SYK with the PH-TH domains of ITK. ITK–SYK consists of 495 amino acids and is smaller compared to both ITK (620 aa) and SYK (635 aa). Another likely consequence of this fusion is loss of the three-dimensional structure responsible for the inactive state conformation of SYK and its switch to an active state. Electron microscopic structure-studies of SYK have shown that it is similar to its homolog ZAP-70 and presumably is in an autoinhibited conformation in which the linker A, C-terminal lobe of kinase domain and linker B come in close contact forming a “linker-kinase sandwich”[13], [18]. It is also important that ITK–SYK, due to loss of the tandem SH2 domains, cannot bind its natural target, the immunoreceptor tyrosine-based activation motifs (ITAMs). The translocation also causes the ITK–SYK fusion-protein to be expressed under the control of the ITK promoter. Similar to other translocation-derived fusion-proteins this means that ITK–SYK always is the result of an abnormal expression both in terms of time and space.

Upon activation, PH-TH domains of ITK and other Tec family members are involved in the translocation of the corresponding proteins to the cell membrane, where they bind to PIP3 (Phosphatidylinositol [3,4,5]-trisphosphate) [19], [20], [21]. The enzyme PI3-kinase generates PIP3, and, moreover, PI3-kinase itself is activated by SYK [22], [23]. SYK has multiple phosphorylatable tyrosine residues involved in a wide array of functions from negative regulation to substrate phosphorylation [24]. Most of the phosphorylatable tyrosine residues in SYK are retained in ITK–SYK, except for Y131 and Y296. Constitutively active SYK is phosphorylated at one of these, namely 352, which corresponds to position 212 of ITK–SYK and is able to carry out downstream signaling with mutated activation-loop paired, conserved tyrosines 525/526, which correspond to the tyrosines 385/386 in ITK–SYK [25], [26].

Activated SYK is known to phosphorylate several downstream signaling molecules, including the adapter protein BLNK [27]. BLNK upon phosphorylation provides docking sites for other crucial signaling components like PLCγ, Vav, and Btk. Binding of these proteins to BLNK results in the formation of a signalosome, which ultimately leads to the activation of specific transcription factors required to modulate cell structure/function. BLNK does not seem to play any major role in T lymphocytes, where instead the adapter protein SLP-76 is crucial for development [28]. This protein is expressed throughout hematopoietic compartment, and SLP-76 loss-of-function blocks T lineage development at the double-negative stage 3 [29], [30].

While this work was in progress a related report was recently published [11], demonstrating that the fusion-protein resulting from this translocation is constitutively active, and is dependent on its PH-domain and displays transformation potential in the NIH-3T3 cell line [11]. These findings nicely complement the results presented in our report, collectively providing a more complete functional picture of the ITK–SYK fusion.