Proteins, introduction phosphorylation

Phosphorylation on serine, threonine, and tyrosine residues is an extremely important modulator of protein function. Phosphorylation can be analyzed by mass spectrometry with enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopep-tides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags (McLachlin and Chait 2001). 

Akt activity is induced in a PI-3K-dependent manner immediately suggesting that the phosphorylated lipid products of PI-3K mediate the activation. Incubation of purified Akt with purified 3-phosphorylated phospholipids results in various extents of activation (44,46). These lipids, such as PtdlnsJP, PtdIns(3,4)P2> and PtdIns(3,4,5)P3, specifically associate with PH domains in a number of proteins (47). Furthermore, co-transfection of a dominant-negative form of PI-3K (delta-p85) also inhibits Akt activation (43). It was later shown that introduction of constitutively active mutants of the catalytic subunit of PI-3K was sufficient to activate Akt in cells (46,48). These studies strongly implicate Akt as a downstream effector of growth-factor-stimulated PI-3K activation in a variety of cell types. 

CONTENTS Acknowledgments, Margery G. Ord and Lloyd A. Stocken. Introduction. Biochemistry Before 1900. Early Metabolic Studies Energy Needs and the Composition of the Diet. Carbohydrate Utilization Glycolysis and Related Activities. Aspects of Carbohydrate Oxidation, Electron Transfer, and Oxidative Phosphorylation. Amino Acid Catabolism in Animals. The Utilization of Fatty Acids. The Impact of Isotopes 1925-1965. Biochemistry and the Cell. Concepts of protein Structure and Function. Chronological Summary of Main Events Up to ca. 1960. Principal Metabolic Pathways. Index. 

As mentioned in the introduction, CaMKKs can phosphorylate proteins in addition to CaMKI and CaMKIV. One such alternate target is the AMP-dependent protein kinase (AMPK) (Hawley et al., 1995 Hamilton et al., 2002), and recent studies present compelling evidence that CaMKKs are physiologically relevant activators of AMPK in cultured cells. These studies are of particular interest as AMPK is a kinase intimately involved in the regulation of metabolism, both at the cellular and organismal levels (Kemp et al., 1999). In mammals, AMPK has been implicated in diabetes, obesity and cardiovascular disease (Arad et al., 2002 Kemp et al., 2003) prompting a flurry of studies to identify the relevant AMPKK involved in each tissue that participates in the etiology of these diseases. 

Signal transduction involves a transfer of information from cell surface receptors to the cytoplasm and ultimately to the nucleus where cellular activation and response are initated (1). Protein-tyrosine kinases (PTKs) serve central roles in many of these pathways by phosphorylating tyrosyl residues, which result in the introduction of phosphotyrosyl (pTyr, 1, Fig. 1) pharmacophores... 

Since the introduction of metal-ion affinity sorbents for the fractionation of proteins [1], the method became popular for the purification of a wide variety of biomolecules. Metal-ion affinity sorbents are also widely used for the immobilization of enzymes. At present, IMAC is a powerful method for separation of phosphorylated macromolecules, particularly proteins and peptides. The significance of techniques for separation and characterization of phosphorylated biomolecules is now increasing, because phosphorylation modulates enzyme activities and mediates cell membrane permeability, molecular transport, and secretion. Phosphorylated peptides can be separated from a peptide mixture on IDA-Sepharose with Fe " ions (Fig. 2). The majority of peptides pass freely through an IMAC column, whereas acidic peptides, including phosphorylated ones, are retained and can be released by a pH gradient. 

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