Epidermal growth factor stimulates translocation of the class II phosphoinositide 3-kinase PI3K-C2β to the nucleus

2009 ◽  
Vol 422 (1) ◽  
pp. 53-60 ◽  
Author(s):  
Hrvoje Banfic ◽  
Dora Visnjic ◽  
Nikica Mise ◽  
Sanjeevi Balakrishnan ◽  
Simona Deplano ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Class Ii ◽  
Kinase Assay ◽  
C2 Domain ◽  
Protein Fusion ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase

Although the class II phosphoinositide 3-kinase enzymes PI3K-C2α and PI3K-C2β act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2β translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2β resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2β co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2β levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2β demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2β in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain, indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2β into the nucleus.

scholarly journals Recruitment of the Class II Phosphoinositide 3-Kinase C2β to the Epidermal Growth Factor Receptor: Role of Grb2

2001 ◽  
Vol 21 (19) ◽  
pp. 6660-6667 ◽  
Author(s):  
Matthew Wheeler ◽  
Jan Domin
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Class Ii ◽  
Rich Region ◽  
Cell Lysates ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase ◽  
Proline Rich Region

ABSTRACT Previously we demonstrated that the class II phosphoinositide 3-kinase C2β (PI3K-C2β) is rapidly recruited to a phosphotyrosine signaling complex containing the activated receptor for epidermal growth factor (EGF). Although this association was shown to be dependent upon specific phosphotyrosine residues present on the EGF receptor, the underlying mechanism remained unclear. In this study the interaction between PI3K-C2β and the EGF receptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the PI3K. Further, a series of N-terminal PI3K-C2β fragments, truncated prior to each proline-rich region, bound the receptor with decreased efficiency. A single proline-rich region was unable to mediate receptor association. Finally, an equivalent N-terminal fragment of PI3K-C2α that lacks similar proline-rich motifs was unable to affinity purify the activated EGF receptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2β is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2β(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2β in vitro, and this effect was reproduced using either SH3 domain expressed as a glutathione S-transferase (GST) fusion. Interaction with Grb2 dramatically increased the catalytic activity of this PI3K. The relevance of this association was confirmed when PI3K-C2β was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2β was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2β mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

scholarly journals A Dual Phosphoinositide-3-Kinase α/mTOR Inhibitor Cooperates with Blockade of Epidermal Growth Factor Receptor in PTEN-Mutant Glioma

2007 ◽  
Vol 67 (17) ◽  
pp. 7960-7965 ◽  
Author(s):  
Qi-Wen Fan ◽  
Christine K. Cheng ◽  
Theodore P. Nicolaides ◽  
Christopher S. Hackett ◽  
Zachary A. Knight ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Mtor Inhibitor ◽  
Growth Factor Receptor ◽  
Epidermal Growth ◽  
Phosphoinositide 3 Kinase

An epidermal growth factor receptor/ret chimera generates mitogenic and transforming signals: evidence for a ret-specific signaling pathway

1994 ◽  
Vol 14 (1) ◽  
pp. 663-675
Author(s):  
M Santoro ◽  
W T Wong ◽  
P Aroca ◽  
E Santos ◽  
B Matoskova ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Egf Receptor ◽  
Biological Effects ◽  
Ectopic Expression ◽  
Growth Factor Receptor ◽  
Dependent Signal ◽  
Epidermal Growth

A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.

scholarly journals Epidermal growth factor (EGF) stimulates association and kinase activity of Raf-1 with the EGF receptor.

1991 ◽  
Vol 11 (2) ◽  
pp. 913-919 ◽  
Author(s):  
H App ◽  
R Hazan ◽  
A Zilberstein ◽  
A Ullrich ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Kinase Assay ◽  
Downstream Effector ◽  
Epidermal Growth

Raf-1 serine- and threonine-specific protein kinase is transiently activated in cells expressing the epidermal growth factor (EGF) receptor upon treatment with EGF. The stimulated EGF receptor coimmunoprecipitates with Raf-1 kinase and mediates protein kinase C-independent phosphorylation of Raf-1 on serine residues. Hyperphosphorylated Raf-1 has lower mobility on sodium dodecyl sulfate gels and has sixfold-increased activity in immunocomplex kinase assay with histone H1 or Raf-1 sequence-derived peptide as a substrate. Raf-1 activation requires kinase-active EGF receptor; a point mutant lacking tyrosine kinase activity in inactive in Raf-1 coupling and association. It is noteworthy that tyrosine phosphorylation of c-Raf-1 induced by EGF was not detected in these cells. These observations suggest that Raf-1 kinase may act as an important downstream effector of EGF signal transduction.

scholarly journals Laminin γ3 Chain Binds to Nidogen and Is Located in Murine Basement Membranes

2005 ◽  
Vol 280 (23) ◽  
pp. 22146-22153 ◽  
Author(s):  
Nikolaus Gersdorff ◽  
Eddie Kohfeldt ◽  
Takako Sasaki ◽  
Rupert Timpl ◽  
Nicolai Miosge
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Basement Membranes ◽  
Electron Microscopic ◽  
Immunogold Staining ◽  
Adult Mice ◽  
Epidermal Growth

Recently a novel laminin γ3 chain was identified in mouse and human and shown to have the same modular structure as the laminin γ1 chain. We expressed two fragments of the γ3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3–5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The γ3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the β2 VI/V fragment. The γ3 III3–5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the γ1 III3–5 fragment. These data suggested that laminins containing the γ3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against γ3 VI/V and γ3 III3–5 showed no cross-reaction with homologous fragments from the γ1 and γ2 chains of laminin and allowed the establishment of γ chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20–100-fold lower content of the γ3 chain compared with the γ1 chain in various tissue extracts of adult mice. The expression of γ3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the γ3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the γ3 chain to basement membranes of these tissues.

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