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Figure 4.
Figure 4 Phosphoinositide-binding properties of wild type and
mutant PDK1. (A) Representation of the Ins(1,3,4,5)P[4]-binding
pocket on the PDK1 PH domain. The residues that make contacts
with Ins(1,3,4,5)P[4] and that are mutated in this study are
labelled. (B) The ability of the indicated wild type and mutant
forms of PDK1 to interact with phosphoinositides was analysed
using a protein-lipid overlay assay. Serial dilutions of the
indicated phosphoinositides (250, 100, 50, 25, 12.5, 6.3, 3.1
and 1.6 pmol) were spotted onto nitrocellulose membranes, which
were then incubated with the purified GST-PDK1 species. The
membranes were washed, and the GST-PDK1 bound to the membrane by
virtue of their interaction with lipid was detected using a GST
antibody (Dowler et al, 2002). A representative of at least two
separate experiments is shown. (*) indicates a long exposure of
the film to detect weak binding.
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