The best way to monitor whether or not the PI3-K kinase seems to be the activation of PKB. In order to be activated, Phosphokinase B (also known as Akt-1) must be phosphorylated on two amino acid residues: Thr 308 which lies on the actual activation segment of the protein and Ser 473 which lies in the hydrophobic motif of the protein. Thr 308 is phosphorylated by PDK-1 when PKB comes in contact with PDK-1 after binding to the Pleckstrine domain of PIP3. Ser 473 must be phosphorylated by a distinct but unstudied kinase PDK-2. By using x-ray crystalography, it is posible to determine whether PKB is inactive due to an unphosphorylated Thr 308 or an unphosphorylated Ser 473.
Figure 14
These pictures demonstrate the activation of PKB under the influences of Caffeine and Theophylline.
Under normal conditions, when insulin binds to it's receptor, the receptor would autophosphorylate itself and would in turn phosphorylate the IRS-1 enzyme. This enzyme would activate PI3-K which could then transform PIP2 to PIP3. With PIP3 present, PKB and PDK-1 could come together and PKB to phosphorylate PKB's Thr 308 residue. While the mechanism and timing of PDK-2 is unknown, phosphorylation of the Ser 473 residue would occur at this time as well. It is clear from this description why the level of activated PKB would be a good indicator of PI3-K activity. This form of measurement was used to monitor the effects of caffeine and theophylline on PI3-K activity. top
Figure 15
The Insulin Pathway
Even under conditions where insuline was available and bound to the insulin receptors, the CHO-IR cells expressed unactive Phosophokinase B under environmental conditions of .5 mM theophylline concentrations and 1 mM concentrations of caffeine. Under scrutiny by X-ray crystalography, it was found that phosphorylation of Thr 308 and Ser 473 were inhibited with equal potency.
Further investigation was conducted in intact rat soleus muscle to ensure that results were not dependant upon culture conditions and the same affects were observed.
Figure 16
Insulin stimulated PKB activity