Introduction

1.1.1 Reversible Covalent Protein Modification

Enzyme-catalyzed reversible covalent protein modification is a widespread cellular event, which effects that the number of individually distinct human protein species is at least one order of magnitude in excess of the number of gene-encoded proteins. Reversible protein phosphorylation catalysed by kinases and phosphatases is probably the most abundant and most important protein modification principle. In addition, several other protein modifying/ demodifying systems are found (e.g. the pairs acetylase/deacetylase, N-methyltransferase/N-demethylase). Side-by-side to the principle of covalent modification, the functional diversity of the proteome is further expanded by the formation of noncovalent complexes exhibiting a unique functionality, activity or cellular localization. Proteins may form homo- and heteromeric complexes as well as noncovalent complexes with metabolites, drugs or metals. The lifetime of a noncovalent protein complex, its localization and its function may be influenced by covalent modifications. Thus, the interplay of covalent modification and noncovalent interaction forms the basis for the eclectic abilities of the proteome as sensor and regulator element in cells. Figure 1.1 gives a graphic summary of this situation.

Annotation of the human genome resulted in the discovery 518 protein kinase and 214 protein phosphatase genes. This means that about 3% of our genome-encoded proteins are functionally specialized for adding a phosphate group to or removing it from proteins including the kinases themselves. As a result, it is estimated that about 30% of the human proteome is phosphorylated in some way. In addition experimental data allow the estimation that phosphoproteins are on average phosphorylated at three sites. Therefore, protein phosphorylation is probably the most abundant reversible protein modification and it is probably the functionally most diverse and influential one.

1.1.2 The Phosphorus Content of a Cell

Phosphorus is a relatively rare element, which occurs in the biosphere mainly as phosphate. Due to its restricted availability and its multiple biochemical functions, phosphate is often a growth-limiting factor. A eukaryotic cell on average contains 0.5 pmol of phosphorus, mainly in the form of organic phosphate. Its distribution over the most important compound classes of a cell is schematically displayed in Figure 1.2.

Lipids contain about one third of the phosphorus content of a cell followed by roughly equal portions (of around 20%) located in metabolites, RNA, and proteins. ATP/ADP and DNA each contain about 5% of total cellular phosphorus, whereas inorganic phosphate (Pi) represents only about 2%.

1.1.3 Nutritional Proteins Recognized as Phosphoproteins

The phosphate content of proteins first became evident in the characterization of acidic nutritional proteins such as casein and vitellin, which at that time were named conjugated proteins. The next progress occurred when phosphoserine was identified as component of vitellin, a protein isolated from egg yolk. The nutritional protein casein is highly phosphorylated, which confers calcium-binding properties to it. In this way both calcium and phosphate are provided for bone formation. The main constituent of bone is calcium hydroxyapatite, which is a mixed calcium hydroxide phosphate. The recognition of nutritional phosphoproteins as sources for phosphate and calcium describes the first known function of phosphorylated proteins.

1.1.4 Protein Phosphorylation Controls Metabolic Enzymes

The efforts of Carl and Gerti Cori in the 1940's, and the subsequent studies of Earl Sutherland, Edwin Krebs and Edmond Fischer paved the way for the revolutionary insight, that phosphate attachment was not only an add-on to nutritional proteins, but may control the function of a key enzyme of energy metabolism: glycogen phosphorylase. This enzyme cuts α-D-glucose from glycogen liberating it as α-D-glucose-1-phosphate by concomitant attachment of inorganic phosphate. The Coris unravelled that glycogen phosphorylase is regulated in its activity by hormones and that it exists in two interconvertible forms, an active and an inactive form. Studying the action of hormones on glycogen metabolism, Sutherland discovered the role of cAMP as the first 'second messenger' in transmembrane signal transduction. Roughly in parallel to these investigations, Krebs and Fischer discovered the principle of reversible phosphorylation as the metabolic control mechanism underlying the activation/deactivation of glycogen phosphorylase. They found that phosphorylation at Ser15 activates glycogen phosphorylase, whereas dephosphorylation led to inactivation. An important analytical step in this discovery was tryptic digestion of glycogen phosphorylase labeled with 32P and isolation of the radiolabeled peptides. The main radiolabeled peptide found was sequenced as KQI-pS-VR. In these years it was also discovered that kinase-catalyzed phosphorylation requires ATP and Mg2+ ions and that kinase-catalyzed reactions can be performed in vitro. Numerous historical and scientific details of this development are summarized in the corresponding Nobel lectures [http:// nobelprize.org], for instance in those of Krebs and Fischer.

1.1.5 Protein Kinase Cascades as Principle of Cellular Signal Transduction

In the structural characterization of kinases two milestones were achieved using cAMP-dependent protein kinase A as model enzyme. It was the first kinase, to be completely sequenced and for which the complete 3D structure was unravelled. As an extension of phosphorylation at serine and threonine, phosphorylation at tyrosine was discovered and viral oncogene products were identified as tyrosine kinases. In the following, a class of receptors with intracellular tyrosine kinase activity was found, laying the ground for unravelling a widespread mechanism for transmembrane signal transduction. In prokaryotes a signal transduction pathway was discovered based on phosphorylation at histidine (phosphoric acid amide) and at aspartate (phosphoric acid ester) (for reviews see e.g.) which represent two chemically more labile forms of phosphate addition compared to the phosphoric acid esters at serine, threonine and tyrosine.

The effect of phosphorylation is amplified, in case a kinase is modified so that its activity is switched on or off. For instance, in a multistep kinase cascades, one kinase activates the next kinase by phosphorylation, so that an exponential amplification of the start event is achieved. Since kinase cascades are the central principle of many signalling cascades, phosphorylation is one of the keys for transformation of extracellular...