Tài liệu Báo cáo khoa học: Receptor association and tyrosine phosphorylation of S6 kinases pdf

Receptor association and tyrosine phosphorylation

of S6 kinases

Heike Rebholz1,2, Ganna Panasyuk3

, Timothy Fenton1,2, Ivan Nemazanyy3

, Taras Valovka4

,

Marc Flajolet5

, Lars Ronnstrand6

, Len Stephens7

, Andrew West7 and Ivan T. Gout2,3

1 Ludwig Institute for Cancer Research, London, UK

2 Department of Biochemistry and Molecular Biology, University College London, UK

3 The Institute of Molecular Biology and Genetics, Kyiv, Ukraine

4 Institute of Veterinary Biochemistry and Molecular Biology, University Zurich, Switzerland

5 Rockefeller University, New York, NY, USA

6 Lund University, Department of Experimental Clinical Chemistry, Malmo, Sweden

7 Babraham Institute, Cambridge, UK

8 GlaxoSmithKline, Harlow, UK

Keywords

AGC kinases; platelet-derived growth factor

receptor; receptor tyrosine kinases;

ribosomal protein S6 kinase; src

Correspondence

H. Rebholz, Box 296, Rockefeller University,

1230 York Ave, New York, NY 10021, USA

Fax: +1 212 327 7888

Tel: +1 212 327 8486

E-mail: [email protected]

(Received 17 August 2005, revised 6

February 2006, accepted 8 March 2006)

doi:10.1111/j.1742-4658.2006.05219.x

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic

stimuli and is a key player in the regulation of cell growth. The activation

process of S6 kinase involves a complex and sequential series of multiple

Ser⁄Thr phosphorylations and is mainly mediated via phosphatidylinositol

3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1)

and mTor-dependent pathways. Upstream regulators of S6K, such as

PDK1 and protein kinase B (PKB⁄Akt), are recruited to the membrane via

their pleckstrin homology (PH) or protein–protein interaction domains.

However, the mechanism of integration of S6K into a multi-enzyme com￾plex around activated receptor tyrosine kinases is not clear. In the present

study, we describe a specific interaction between S6K with receptor tyrosine

kinases, such as platelet-derived growth factor receptor (PDGFR). The

interaction with PDGFR is mediated via the kinase or the kinase extension

domain of S6K. Complex formation is inducible by growth factors and

leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have

shown that the phosphorylation is exerted via a PDGFR-src pathway. Fur￾thermore, src kinase phosphorylates and coimmunoprecipitates with S6K

in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or

src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction

in tyrosine phosphorylation of S6K. In addition, we mapped the sites of

tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively.

Mutational and immunofluorescent analysis indicated that phosphorylation

of S6Ks at these sites does not affect their activity or subcellular localiza￾tion. Our data indicate that S6 kinase is recruited into a complex with

RTKs and src and becomes phosphorylated on tyrosine ⁄s in response to

PDGF or serum.

Abbreviations

btk, Bruton’s tyrosine kinase; CSFR, colony stimulating factor receptor; DBS, donor bovine serum; DMEM, Dulbecco’s modified Eagle’s

medium; FBS, fetal bovine serum; FITC, fluoroscein isothiocyanate; HGFR, hepatocyte-growth factor receptor; PDGF, platelet-derived

growth factor; PDGFR, platelet-derived growth factor receptor; PDK1, 3-phosphoinositide-dependent protein kinase-1; PH, pleckstrin

homology; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKB ⁄ Akt, protein kinase B; PKC, protein kinase

C; PTB, phosphotyrosine binding domain; RTK, receptor tyrosine kinase; S6K, ribosomal protein S6 kinase; SH2, Src homology 2.

FEBS Journal 273 (2006) 2023–2036 ª 2006 The Authors Journal compilation ª 2006 FEBS 2023