Signaling of the ITK (IL2-inducible T-cell kinase) -SYK fusion kinase is dependent on adapter SLP-76 (SH2 domain-containing leukocyte protein of 76 kD) and on the adapter function of the kinases SYK/ZAP70 (zeta-chain [TCR] associated protein kinase 70 kD).
ABSTRACT/SUMMARY ITK-SYK oncogene consists of the TH-PH domain of inducible T cell kinase (ITK) and the kinase domain of spleen tyrosine kinase (SYK), and is believed to be the cause of peripheral T cell lymphoma (PTCL). We and others have recently demonstrated that this fusion protein is constitutively tyrosine phosphorylated and is transforming both in vitro and in vivo. To get 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 herein show 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 was independent of their kinase activity. Notably, ITK-SYK expression in SYF cells phosphorylates SLP-76 in the absence of SRC family kinases (SFKs). Altogether our data suggest that ITK-SYK exists in active conformation state and is therefore capable of signaling without SFKs or stimulation of the TCR.