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Mutation of tyrosine residue 857 in the PDGF β-receptor affects cell proliferation but not migration
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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research. (PDGF signaling)
2010 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 22, no 9, 1363-1368 p.Article in journal (Refereed) Published
Abstract [en]

Activation of platelet-derived growth factor (PDGF) receptors occurs through ligand-induced dimerization and autophosphorylation. In this study, we investigated the effects of mutation of tyrosine residue 857 (Y857) in the activation loop of the PDGF beta-receptor (PDGFR beta) to phenylalanine (Y857F). In agreement with previous observations, we found that PDGFR beta(Y857F) had a severely diminished in vitro kinase activity. However, in vivo the overall amount of tyrosine phosphorylation of PDGFR beta(Y857F) was similar to that of the wild-type receptor, except for the tyrosine residue 771 (Y771) which displayed a stronger phosphorylation in the mutant receptor. Analysis of the ability to induce signal transduction revealed that the PDGFR beta(Y857F) mutant had an attenuated activation of Akt and Erk1/2 MAP kinase. In contrast, the mutant receptor efficiently mediated phosphorylation of the ubiquitin-ligase c-Cbl that participates in receptor internalization and degradation, and PLC gamma which has previously been shown to be connected with various cellular responses, including migration. However, the protein tyrosine phosphatase SHP-2, implicated in the PDGF-induced mitogenic response, together with the adaptor proteins Alix and Stam, involved in intracellular sorting of receptor, was not phosphorylated in cells expressing PDGFR beta(Y857F). We found that both receptor variants were internalized from the cell surface and degraded at a comparable rate. Interestingly, PDGFR beta(Y857F) was unable to mediate PDGF-BB-induced mitogenic signaling, whereas it could elicit a chemotactic response.

Place, publisher, year, edition, pages
2010. Vol. 22, no 9, 1363-1368 p.
Keyword [en]
PDGF; Y857; Autophosphorylation; SHP-2; Proliferation; Chemotaxis
National Category
Cell and Molecular Biology Cell and Molecular Biology Cell and Molecular Biology
Research subject
Biology with specialization in Molecular Cell Biology
Identifiers
URN: urn:nbn:se:uu:diva-122471DOI: 10.1016/j.cellsig.2010.05.004ISI: 000279889300011OAI: oai:DiVA.org:uu-122471DiVA: diva2:310211
Available from: 2010-04-14 Created: 2010-04-13 Last updated: 2017-12-12Bibliographically 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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