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The Fer tyrosine kinase is necessary for platelet-derived growth factor-BB-induced Stat3 phosphorylation and colony formation in soft agar
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research. (PDGF signaling)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research. (PDGF signaling)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research. (PDGF Translational Research Group)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Fer is a cytoplasmic tyrosine kinase that is activated in response to platelet-derived growth factor (PDGF) stimulation. In the present report, we show that Fer associates with the activated PDGF b-receptor (PDGFRb) through multiple autophosphorylation sites, i.e. Tyr579, Tyr581, Tyr740 and Tyr1021. Using low molecular weight inhibitors we found that PDGF-BB-induced Fer activation is dependent on PDGFRb kinase activity, but not on the enzymatic activity of Src or Jak kinases. To elucidate the function of Fer downstream of PDGFRb, we downregulated the expression using siRNA; under conditions with reduced Fer expression, PDGF-BB was unable to induce phosphorylation of Stat3, whereas Stat5, Erk1/2 and Akt were unaffected. In addition, PDGFRb autophosphorylation was partially dependent on Fer expression. On a functional level, we could demonstrate that expression of Fer is dispensable for PDGF-BB-induced proliferation and migration. However, Fer was found to be essential for colony formation in soft agar, consistent with its critical role in Stat3 activation, which has frequently been implicated in cell transformation.

National Category
Biochemistry and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-122467OAI: oai:DiVA.org:uu-122467DiVA: diva2:310210
Available from: 2010-04-14 Created: 2010-04-13 Last updated: 2010-05-12Bibliographically approved
In thesis
1. Regulation of PDGFRβ signaling 
Open this publication in new window or tab >>Regulation of PDGFRβ signaling 
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelet-derived growth factor (PDGF) isoforms, which bind to closely related a- and b-tyrosine kinase receptors, induce migration, proliferation, survival and differentiation of mesenchymal cells. They signal by the active receptor attracting Src homology 2 (SH2) domain containing proteins, which subsequently initiate a set of signaling pathways. The aim of this thesis was to elucidate regulatory mechanisms involved in PDGFRb signaling.

In the first two projects we investigated the roles in downregulation of PDGFRb of two related adaptor proteins, i.e. ALG-2 interacting protein X (Alix) and His-domain containing protein tyrosine phosphatase (HD-PTP) functions of. We found that Alix and HD-PTP influence ubiquitination of PDGFRb following PDGF stimulation, by affecting the E3 ligase c-Cbl. Alix enhances complex formation between c-Cbl and PDGFRb, increases c-Cbl phosphorylation and decreases its stability. Interestingly, while both HD-PTP and Alix participate in degradation of PDGFRb, only Alix affects receptor internalization. Moreover, we demonstrated that absence of HD-PTP promotes cell proliferation. In conclusion, we suggest that both Alix and HD-PTP are important adaptor proteins in regulation of PDGFRb downregulation, although the observed differences between their actions suggest that Alix and HD-PTP exert their functions via different mechanisms.

The third study explored the importance of tyrosine residue 857 in the activation loop of PDGFRb. We report that, in vitro the tyrosine residue 857 to phenylalanine (Y857F) mutant receptor kinase activity is diminished while in vivo it does not affect the phosphorylation of PDGFRb. The phosphorylation pattern of PDGFRb revealed that most sites in the Y857F mutant receptor were phosphorylated similarly as in the wild-type receptor. However, tyrosine residue 771 was found to be hyperphosphorylated in the Y857F mutant receptor. This may be due to defective phosphorylation and activation of SHP-2, since it has been shown to dephosphorylate the receptor at Y771. In addition, activation of the Erk1/2 and Akt pathways was defective downstream of the Y857F mutant receptor. Interestingly, the Y857F mutant receptor was able to mediate cell migration, but not proliferation.

The last study investigated a role of the tyrosine kinase Fer in PDGF signaling. We showed that Fer interacted with and was activated by PDGFRb in a ligand-dependent manner. In cells depleted of Fer, receptor phosphorylation was decreased and phosphorylation of Stat3 was abolished, whereas Stat5, Erk1/2 and Akt were activated normally. Colony formation in soft agar was abolished in cells depleted of Fer, but no effect was seen on cell proliferation and migration. Since Stat3 has been shown to be involved in transformation, we speculate that phosphorylation of Stat3 in Fer-depleted cells, affects the ability of cells to form colonies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 563
Keyword
PDGF, HD-PTP, Alix, Cbl, Ub, Fer, Y857, Y771, downregulation, degradation, ubiquitination, activation loop
National Category
Biochemistry and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-123045 (URN)978-91-554-7813-1 (ISBN)
Public defence
2010-06-02, B42, BMC, BMC, 09:00 (English)
Opponent
Supervisors
Available from: 2010-05-11 Created: 2010-04-22 Last updated: 2010-05-24

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