STAP-2 is phosphorylated at tyrosine-250 by Brk and modulates Brk-mediated STAT3 activation

https://doi.org/10.1016/j.bbrc.2009.04.076Get rights and content

Abstract

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains Pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. STAP-2 is also known as breast tumor kinase (Brk) substrate (BKS). Our previous studies revealed that STAP-2 binds to signal transducer and activator of transcription 3 (STAT3) and STAT5, and regulates the signaling pathways downstream of them. In the present study, we identified tyrosine-250 (Tyr250) in STAP-2 as a major site of phosphorylation by Brk, using a series of STAP-2 YF mutants and anti-phospho-STAP-2 Tyr250 antibody. Furthermore, overexpression of the STAP-2 Y250F mutant protein affected Brk-mediated STAT3 activation. Importantly, small-interfering RNA-mediated reduction of endogenous STAP-2 expression decreased Brk-mediated STAT3 activation. Taken together, our findings demonstrate that STAP-2 is phosphorylated at Tyr250 by Brk, and plays an important role in 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)

There are more references available in the full text version of this article.

Cited by (45)

  • Targeting protein tyrosine kinase 6 in cancer

    2020, Biochimica et Biophysica Acta - Reviews on Cancer
  • STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization

    2017, Journal of Biological Chemistry
    Citation Excerpt :

    STAT3 is activated by not only EGFR but also IL-6R signaling, and blockade of IL-6R inhibits tumor growth, suggesting that STAP-2 knockdown represses prostate tumor growth by synergistic effects of down-regulation of EGFR and IL-6R signaling. Our previous studies showed that STAP-2 associates with BRK and enhances BRK-mediated STAT3 and STAT5 activation (14, 15). BRK is known to up-regulate EGFR signaling by inhibiting c-CBL–mediated EGFR ubiquitination (25).

  • STAP-2 interacts with Pyk2 and enhances Pyk2 activity in T-cells

    2017, Biochemical and Biophysical Research Communications
View all citing articles on Scopus
1

These authors contributed equally to this work.

View full text