STAP-2 is phosphorylated at tyrosine-250 by Brk and modulates Brk-mediated STAT3 activation
Introduction
Protein-tyrosine kinases (PTKs) play critical roles in regulating cell growth, differentiation and transformation. Tyrosine kinases themselves become autophosphorylated within the activation segment of their kinase domains, thereby inducing conversion to a more active state. However, a frequent consequence of tyrosine phosphorylation is the creation of specific binding sites for adaptor proteins that contain Src homology (SH) 2 domains. Such phosphotyrosine-dependent protein–protein interactions serve to recruit regulatory proteins to phosphorylated receptors and other adaptor proteins, and thereby activate signaling pathways that control numerous aspects of cellular functions [1], [2]. The non-receptor tyrosine kinase breast tumor kinase (Brk) was originally isolated from a human breast carcinoma cells [3]. Brk is also known as PTK6, having been identified as a highly expressed PTK in human melanocytes [4], and a cDNA for its mouse homolog, Sik, which has 80% amino acid identity to Brk/PTK6, was cloned from mouse intestinal crypt cells [5]. Brk contains an SH3 domain, an SH2 domain, and a tyrosine kinase catalytic domain, but it lacks an N-terminal myristoylation site for membrane targeting. Subsequent characterization of Brk showed it to be present in approximately 60% of human breast tumors, yet absent in normal or fibrocystic mammary tissues. Brk has also been shown to be expressed in other cancer cells, including metastatic melanomas and colon and prostate tumors [6], [7], [8], [9]. However, the molecular mechanism by which Brk participates in tumorigenesis remains poorly characterized. One substrate of Brk is BKS (Brk substrate)/signal-transducing adaptor protein-2 (STAP-2), which has also been implicated in modulating the activity of STAT3 and STAT5 [10], [11], [12]. STAP-2 was identified as a c-fms-interacting protein, and contains an N-terminal pleckstrin homology (PH) domain and a region distantly related to the SH2 domain [11]. The central region of STAP-2, which is distantly related to the SH2 domain, shares 29% sequence identity with the SH2 domain of human PLCγ2. Furthermore, STAP-2 possesses a C-terminal proline-rich region and a STAT3-binding YXXQ motif [11].
In the present study, we identified tyrosine-250 (Tyr250) as the major site of phosphorylation of STAP-2 by Brk. We also show that the kinase activity of Brk is required for a direct interaction with STAP-2. Furthermore, we demonstrate that a reduction of endogenous STAP-2 expression decreases Brk-mediated STAT3 activation.
Section snippets
Materials and methods
Reagents and antibodies. Expression vectors, STAP-2 and its YF (substitution of Tyr to Phe) mutants were described previously [11]. Expression vectors for wild-type Brk (Brk WT), Brk K219M and STAT3-LUC were provided by Dr. A. Harvey (Brunel University, Middlesex, UK) and Dr. T. Hirano (Osaka University, Osaka, Japan), respectively [3], [13]. Anti-Myc and -GST antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FLAG antibody was obtained from Sigma–Aldrich (St.
Brk phosphorylates STAP-2 at Tyr250
STAP-2/BKS was originally identified as a substrate for Brk [10]. However, the tyrosine residue in STAP-2 that undergoes phosphorylation by Brk remained unknown. In the present study, we attempted to identify the site of Brk-mediated tyrosine phosphorylation in STAP-2. We first confirmed tyrosine phosphorylation of STAP-2 by Brk in vivo. Myc-tagged STAP-2 was expressed without or with FLAG-tagged wild-type Brk (Brk WT) or a kinase inactive form of Brk, Brk K219M, in 293T cells. The cells were
Acknowledgments
This study was supported in part by Grant-in-Aid for scientific research from Ministry of Education, Culture, Sports, Science and Technology of Japan.
References (20)
Specificity in signal transduction: from phopshotyrosine-SH2 domain interaction to complex cellular system
Cell
(2004)- et al.
Protein phosphorylation in signaling-50 years and counting
Trends Biochem. Sci.
(2005) - et al.
STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif
J. Biol. Chem.
(2003) - et al.
Physical and functional interactions between STAP-2/BKS and STAT5
J. Biol. Chem.
(2005) - et al.
Leukemia inhibitory factor-induced phosphorylation of STAP-2 on tyrosine-250 is involved in its STAT3-enhancing activity
Biochem. Biophys. Res. Commun.
(2007) - et al.
Cloning and characterisation of cDNAs encoding a novel non-receptor tyrosine kinase, brk, expressed in human breast tumours
Oncogene
(1994) - et al.
A survey of protein tyrosine kinase mRNAs expressed in normal human melanocytes
Oncogene
(1993) - et al.
Tyrosine kinase gene expression in the mouse small intestine
Oncogene
(1994) - et al.
BRK tyrosine kinase expression in a high proportion of human breast carcinomas
Oncogene
(1997) - et al.
Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma
Int. J. Cancer
(1997)
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These authors contributed equally to this work.