Ser/Thr specific protein kinase

Adenylate cyclase was identified as the primary Ras target in yeast (Saccha-romyces cerevisiae) [56] but it took a while before in 1993 several groups independently found Raf to be the effector of Ras in mammals [41-44]. Shortly afterwards it was realized that this is not the only target of Ras but up until now it appears to be the most prominent one. Raf is a Ser/Thr-specific protein kinase which phosphorylates and thereby activates Mek which in turn phosphorylates and activates Erk, leading to an amplification of the signal. Erk, also termed MAPK, has a plethora of phosphorylation targets, the most important of which are transcription factors such as Elk-1, leading to activation of the transcription machinery in the nucleus. 

The progression of the cell cycle is regulated by interconversion processes, in each phase, special Ser/Thr-specific protein kinases are formed, which are known as cyclin-depen-dent kinases (CDKs). This term is used because they have to bind an activator protein (cyclin) in order to become active. At each control point in the cycle, specific CDKs associate with equally phase-specific cyclins. if there are no problems (e.g., DNA damage), the CDK-cyclin complex is activated by phosphorylation and/or dephosphorylation. The activated complex in turn phosphorylates transcription factors, which finally lead to the formation of the proteins that are required in the cell cycle phase concerned (enzymes, cytoskeleton components, other CDKs, and cyclins). The activity of the CDK-cyclin complex is then terminated again by proteolytic cyclin degradation. 

Classification, Structure and Characteristics of Ser/Thr-specific Protein Kinases... 

Hie first protein kinase obtained in a purified form was the Ser/Thr-specific phospho-rylase kinase of muscle, in 1959 (Krebs et al., 1959). With the discovery of the Tyr-specific protein kinases (Erikson et al., 1979), the Ser/Thr-specific protein kinases were joined by another extensive class of protein kinases of regulatory importance, to which a central function in growth and differentiation processes was soon attributed. At present, several hrmdred different protein kinases are known in mammals, most of which are Ser/Thr- or Tyr-specific. In addition, there are some protein kinases that phospho-rylate other amino acids (review Hrmter, 1991). 

Ser/Thr-specific protein kinases esterify a phosphate residue with the alcohol group of Ser and Thr residues. 

Due to the complexity and number of the subfamilies, only selected subfamilies of the Ser/Thr-specific protein kinases in vertebrates are presented in the following (according to Himter, 1991). 

Structural information on Ser/Thr-specific protein kinases (review Goldsmith and Cobb, 1994, Johnson et al., 1996, Johnson, 1998) indicates a markedly conserved structure of the catalytic domain. In Fig. 7.2a, the structure of the catalytic subimit of protein kinase A is shown in complex with an inhibitor peptide (Knighton et al., 1991). In this case, the inhibitor peptide serves as a model for a phosphorylation substrate. In the form of two Arg residues, it possesses part of a sequence characteristic of phosphorylation sites of protein kinase A, which is defined by two Arg residues in the neighborhood of the Ser residue to be phosphorylated (Fig. 7.3). The inhibitor peptide lacks the... 

Structural information on autoinhibition is available for the twitchin kinase. The twitchin kinase is a Ser/Thr-specific protein kinase of the nematode Caenorhabditis ele-gans and is homologous to the myosin light chain kinase of mammals (see 7.4.1). The crystal structure of a catalytic fragment of twitchin kinase (Hu et al., 1994) has an auto-inhibitory element at the C-terminus, which makes specific contact with parts of the active site and the ATP binding site. The active site of twitchin kinase is blocked by the autoinhibitory structural element by ... 

TTie family of protein kinase C enzymes includes Ser/Thr-specific protein kinases that require the following cofactors for activation (review articles Dekker and Parker, 1994 Newton, 1997, Oancea and Meyer, 1998) ... 

Like most of the Ser/Thr-specific protein kinase family, the protein kinase C family also shows significant heterogeneity. At the present time, at least 12 different subtypes of protein kinase C have been discovered in mammals, based on different criteria such... 

The current state of Ser/Thr phosphorylation of a protein is determined by the relative activity of Ser/Thr-specific protein kinase and protein phosphatase. It is therefore imderstandable that the cell has had to develop special mechanisms to balance the two activities with one another, and, when needed, to allow kinase or phosphatase activity to dominate. One of the best investigated examples of coordinated activity of protein kinases and protein phosphatases is the regulation of glycogen metabolism in skeletal muscle. Glycogen metabolism is an example of how two different signals, namely a cAMP signal and a Ca signal meet in one metabolic pathway and control the activity of one and the same enzyme. 

Transforming growth factor-P, TGF-P1,-P2, -P3 Homodimer of 25 kD TGFP receptor I und II, contains Ser/Thr specific protein kinase activity... 

Fig. 8.7. Structure of the catalytic domain of the insulin receptor. The crystal structure of the tyrosine kinase domain of the insulin receptor (Hubbard et al., 1994) has a two-lobe structure that is very similar to the structure of the Ser/Thr-specific protein kinases. Structural elements of catalytic and regulatory importance are shown. The P loop mediates binding of the phosphate residue of ATP the catalytic loop contains a catalytically essential Asp and Asn residue, found in equivalent positions as conserved residues in many Ser/Thr-specific and Tyr-specific protein kinases. Access to the active center is blocked by a regulatory loop containing three Tyr residues (Tyrll58, Tyrll62 and Tyrll63). Tyrll62 undergoes autophosphorylation in the course of activation of the insulin receptor. MOLSKRIPT representation according to Kraulis, (1991).

The pleckstrin homology (PH) domain is a structural motif of ca. 100 amino acids foimd in many signal molecules such as Ser/Thr-specific protein kinases, tyrosine kinases, isoforms of phospholipase C (PL-CP, y and 6), in G nucleotide exchange factors, adaptor proteins, and in proteins of the cytoskeleton (see also Fig. 8.10). Originally, the PH domain was foimd in the 47 kDa pleckstrin protein, which is the main substrate of protein kinase C in platelets. 

The extent of tyrosine phosphorylation of signal proteins is determined both by the activity of the tyrosine kinases and also the activity of tyrosine-specific protein phosphatases. If the total activity of both enzymes in the cell is considered, it is found that there is a preponderance of protein tyrosine phosphatase activity compared to tyrosine kinase activity. In contrast, the activities of the Ser/Thr-specific protein kinases and protein phosphatases are approximately balanced. It is estimated that the activity of the protein tyrosine phosphatases is about 3-4 orders of magnitude higher than the activity of the protein tyrosine kinases. With this relationship between the activities, it is not surprising that the net level of tyrosine phosphorylation in the cell is very low and that tyrosine phosphorylation is often only transient. Consequently, it took a relatively long time until the importance of tyrosine phosphorylation for signal transduction was assessed correctly. 

Another mechanism of regulation of protein tyrosine phosphatases is via Ser/Thr phosphorylation. Specific phosphorylation of protein tyrosine phosphatases by Ser/ Thr-specific protein kinases of types A and C has been reported (see Neel and Tonks, 1997). This observation indicates the possibility that signal transductions via Ser/Thr kinases and via Tyr kinases/phosphatases may cooperate and that different signal pathways may be crosslinked in this way. 

The MEK kinases (MAPKK kinases) are Ser/Thr-specific protein kinases and are the entry point for signal transduction in a MAPK module. The best characterized representative, Raf-1 kinase, is activated by Ras protein in its GTP-bound form. Raf kinase phosphorylates downstream MEK proteins at two Ser residues, which are separated by three other amino acids. All known MEK proteins have a similar phosphorylation site in the conserved sequence LID/NSXANS/T (X any amino acid). Other representatives of the MEK kinase group are Mos kinase and the protein kinases MEKKl—3. 

Activation of Ser/Thr-specific proteins kinases such as the integrin-linked kinase (ILK)... 

A biochemical system is at the center of the cell cycle, of which the most important players are Ser/Thr-specific protein kinases and regulatory proteins associated with these. The activity of this central cell cycle apparatus regulates processes downstream that help to carry out the many phase-specific biochemical reactions of the cell cycle in a defined order. 

The activity of Ser/Thr-specific protein kinases is often controlled by autoinhibitory sequences (see 7.1.5). Loss or lack of function of autoregulatory sequences due to an oncogenic mutation can remove Ser/Hir kinase activity bound into mitogenic signaling pathways from normal control and thereby promote tumors. An example is the Raf kinase (see 9.6). Viral v-Raf oncoproteins are characterized by a deletion of the NH2-terminal regulatory sequences. 

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