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Mitina, Olga (2005): Src kinases and Flt3: phosphorylation, interference with receptor maturation and mechanism of association. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

Flt3 is the most recently discovered member of the platelet-derived growth factor receptor (PDGFR) subfamily of receptor tyrosine kinases (RTKs). It is expressed on early hematopoietic progenitor cells and is involved in their growth. Activating mutations of Flt3, such as internal tandem duplications (ITD) in the juxtamembrane (JM) region of Flt3, are among the most common genetic alterations found in patients with acute myeloid leukemia (AML). For this reason Flt3 is an attractive target for specific tyrosine kinase inhibitors, and a number of such compounds are currently being tested in clinical trials. However, the mechanisms of Flt3-mediated signal transduction are poorly understood. Src family tyrosine kinases (SFKs) are involved in the signaling processes of various RTKs, including those of the PDGFR subfamily . Most SFKs are expressed predominantly or exclusively in hematopoietic cells, and at least Hck and Lyn were found to be activated in AML patients. These observations indicate that SFKs could also be involved in Flt3 signaling. In this work the role of SFKs in signal transduction of the Flt3 receptor was investigated. A panel of Hck and Flt3 mutants was used in biochemical and biological assays to deduce the contribution of kinase activity, SH2 and SH3 binding domains and tyrosine phosphorylation status to Flt3-SFK interaction. The work presented shows that SFKs interfere with the maturation of wild type and Flt3 ITD receptors in human embryonic kidney 293 (HEK- 293) cells in a kinase-dependent manner. The SFK-mediated effect on receptor maturation is reflected by accumulation of the immature, intracellular form of Flt3 at the expense of the mature, plasma membrane-inserted form of the receptor. This effect of SFK kinase activity on receptor maturation is not limited to Flt3, because analogous results were also obtained for the Kit receptor, another member of the PDGFR subfamily. The demonstration of the role of SFKs in regulation of receptor maturation is novel and the exact mechanism underlying this effect requires further investigation. Using the HEK-293 cells it was also shown that Hck is able to phosphorylate Flt3 on tyrosine residues and to associate with autophosphorylated Flt3 in an SH2 domain-dependent manner. Hck-mediated tyrosine phosphorylation occurs in the JM region of Flt3. Tyrosine residues 589 and 591 in the JM region, when phosphorylated, were also identified as the docking sites for Hck. Although Hck phosphorylation and binding sites on Flt3 overlap, Hck-mediated phosphorylation of Flt3 is not sufficient for Flt3-Hck association. In contrast to the situation in HEK-293 cells, Hck neither interferes with Flt3 maturation nor phosphorylates Flt3 on tyrosine residues, and does not detectably associate with the Flt3 receptor in the hematopoietic murine cell line 32D clone 3 (32D cl.3). However, the level of the ectopic expression of Hck in 32D cl.3 cells was lower than in HEK-293 cells. This difference in Hck expression level probably accounts for both the lack of the Hck-mediated phosphorylation of Flt3 and the interference with its maturation in 32D cl.3 cells. The lower Hck expression level, however, does not account for the lack of detectable association of Hck with Flt3 in 32D cl.3 cells. Using biological assays in which activation of Flt3 can partially overcome the requirement for cytokine stimulation in 32D cl.3 survival and proliferation, it was shown that Hck is not involved in Flt3 signal transduction leading to Flt3-mediated cell survival and Flt3 ITD-dependent cell growth. Therefore, biological relevance of SFKs in Flt3 signaling remains unclear. In summary, although the binding and phosphorylation of Flt3 by Hck can be shown, the biological relevance of Hck in Flt3 signaling remains to be formally demonstrated. This research led to the novel finding that, at least when over-expressed or hyperactivated, SFKs interfere with the maturation process of RTKs. Various studies have shown that the ligandindependent activation of RTKs as well as their premature phosphorylation can interfere with maturation of RTKs. This study reveals that, by virtue of their phosphorylating of Flt3, Kit and possibly other RTKs, SFKs can regulate maturation of these RTKs and consequently alter their transport to the plasma membrane. Further studies are required to investigate the biological relevance of this function of SFKs.