Akt/protein kinase B (PKB) is a serine/threonine kinase that mediates many of the anabolic actions of insulin (1), as well as the growth-promoting and/or anti-apoptotic effects of other growth factors, cytokines, or transforming oncogenes (2). These agonists all stimulate Akt/PKB by promoting its phosphorylation on two regulatory residues (e.g., S473 and T308 for the Akt1 isoform), an event dependent on the prior activation of a signaling pathway initiated by the lipid kinase phosphatidylinositol 3-kinase. Once Akt/PKB is activated, it phosphory-lates numerous different substrates, including transcription factors (e.g., FKHR1, others), anti-apoptotic enzymes (e.g., Bad, caspase-9), and metabolic enzymes (e.g., glycogen synthase kinase 3 b), to regulate this diverse array of biological processes. Herein we describe a method for measuring the catalytic activity of Akt/PKB isolated from cell or tissue extracts by quantifying its ability to catalyze phosphate incorporation into an exogenous substrate (outlined in Fig. 1). This chapter illustrates techniques for immunoprecipitating Akt/PKB, choosing an appropriate substrate for the reaction, and optimizing the assay conditions.

Although recombinant Akt/PKB can be prepared using baculovirus (3), most researchers are interested in measuring the activity of Akt/PKB in tissues or cultured cells. Akt/PKB is typically isolated from these tissues by immunoprecip-itation, and a variety of antibodies raised against the Akt/PKB regulatory domain can be used to extract Akt/PKB from cell and tissue lysates without affecting its catalytic activity. Alternatively, many researchers choose to evaluate the activity of wild-type or mutant forms of Akt/PKB that have been over-expressed in different cell lines. For example, Akt/PKB can be epitope-tagged

Fig. 1. Schematic diagram depicting the major steps in the Akt/PKB kinase assay.

on either its amino or carboxyl terminus and can then be immunoprecipitated with antibodies recognizing the epitope (4,5).

Akt/PKB phosphorylates a wide variety of cellular proteins in vivo and can phosphorylate histone H2B and several engineered peptides in vitro. The choice of substrate for the Akt/PKB kinase reactions largely depends on the method to be used for detecting phosphate incorporation. One can monitor in vitro Akt kinase activity by quantifying the amount of radioactive phosphate transferred from [y-32P]ATP into the substrate protein or, alternatively, by immunodetect-ing newly phosphorylated Akt substrates with phospho-specific antibodies. The radioactive method will work with multiple different substrates. Histone-2B (H2B) was one of the first proteins used to measure Akt/PKB's catalytic activity and is used frequently because it is commercially available and relatively inexpensive. However, H2B is not exclusively phosphorylated by Akt/PKB, which could prove problematic should contaminants, such as cAMP- or cGMP-activated protein kinases, coprecipitate alongside Akt/PKB. As will be shown, more specific substrates, such as glycogen synthase kinase-3b (GSK3P), work equally well in the radioactive assay (see Fig. 2). Alternatively, if using GSK3p or Bad as a substrate, one can detect the phosphorylated product using commercially available antibodies recognizing the phosphorylated form. Immun-odetection with a phospho-specific antibody precludes the necessity of using radioactive ATP for detecting phosphate incorporation. Regardless of which substrate is used, sufficient quantities must be added to keep the reaction in the linear range of enzyme activity. Under the conditions to be described, most of the substrate can be phosphorylated within 10 min of the addition of ATP.

Wherever possible, the in vitro assay described herein should be complemented with studies assessing Akt/PKB regulation and/or activity in vivo. Specifically, the phosphorylation state of Akt/PKB can be assessed by immunoblotting cell lysates with phospho-specific antibodies against either regulatory residue (antibodies available from New England Biolabs, Beverly, MA, Biosource International, Camarillo, CA, and others). Alternatively, the activity of Akt/PKB can be assessed by immunoblotting cell lysates with phospho-specific antibodies that recognize various Akt substrates (e.g., phospho-BAD, phospho-GSK3b, and phospho-Akt-substrate panel antibodies from New England Biolabs).

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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