LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

from our laboratory demonstrated that a motor protein required for intracellular transport,
the kinesin 1 heavy chain protein KIF5B, formed a complex with 14-3-3γ in a proteomic
screen (Amitabha Mukhopadhaya unpublished data). Kinesin 1 motor proteins transport
cargos along microtubules. A kinesin motor generally consists of a kinesin motor domain,
which is conserved among kinesin superfamily proteins (KIFs), and unique stalk and tail
domains that are used for kinesin dimerization and/or kinesin binding to cargos, adaptors
or scaffold proteins. The kinesin motor domain generates force by hydrolysing ATP (45).
In some cases, adaptors and scaffolds provide a mechanistic link between kinesins and
cargos, for the recognition of specific cargos and the regulation of cargo loading and
unloading.
As KIF5B potentially formed a complex with 14-3-3γ, immunoprecipatiation
experiments were performed to confirm 14-3-3γ forms a complex with KIF5B. HCT116
cells were transfectd with either HA tagged 14-3-3γ constructs or vector control and
immunoprecipitations performed with antibodies against the HA epitope as described
(353) followed by Western blotting with antibodies specific to KIF5B. 14-3-3γ was able
to form a complex with KIF5B in these assays (Figure 4.3.20 B). These results suggest
that 14-3-3γ can bind to KIF5B.
Figure 4.3.20: 14-3-3γ interacts with PG and Kinesin 1 motor protein KIF5B. (A)
Protein extracts prepared from HCT116 cells were incbubated with either GST or GST-
14-3-3γ on glutathione Sepharose beads. The reactions were resolved on 10% SDS
PAGE gels followed by Western blot analysis with antibodies against plakoglobin. The
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