Src kinases and Flt3: phosphorylation, interference with receptor maturation and mechanism of association

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.