Metabolic enzymes protein kinases

The extent and specificity of the reactions of protein kinases and protein phosphatases are extremely dependent on the degree to which substrate and enzyme are localized at the same place in the cell. Many substrates of protein kinases occur either as membrane associated or particle associated forms (see 7.6.1, enzymes of glycogen metabolism). For protein kinases or protein phosphatases to perform their physiological function in a signal transduction process, they must be transported to the location of then-substrate in many cases (review Hubbard and Cohen, 1992 Mochly-Rosen, 1995). This is vahd both for the Ser/Tbr-specific protein kinases as well as for many Tyr-speci-fic protein kinases. In the course of activation of signal transduction pathways, com-partmentahzation of protein kinases, redistributed to new subcellular locations, is often observed. 

Am. The hormones epinephrine and glucagon cannot penetrate cell membranes. They affect metabolic processes by binding to specific receptors on the membrane, which receptors in turn activate a specific enzyme bound to the inner membrane surface, adenylate cyclase. This enzyme converts ATP to cyclic AMP (cyclic adenosine monophosphate), or c-AMP. The presence of c-AMP activates another enzyme, protein kinase, which phosphorylates and activates phosphorylase kinase. Phosphorylase kinase phosphorylates phosphorylase b (inactive) to form phosphorylase a (active) which in turn cleaves glucose from glycogen by phosphorolysis to yield glucose-I-PO4. 

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

Protein kinase A (PKA) is a cyclic AMP-dependent protein kinase, a member of a family of protein kinases that are activated by binding of cAMP to their two regulatory subunits, which results in the release of two active catalytic subunits. Targets of PKA include L-type calcium channels (the relevant subunit and site of phosphorylation is still uncertain), phospholam-ban (the regulator of the sarcoplasmic calcium ATPase, SERCA) and key enzymes of glucose and lipid metabolism. 

The affect of Li+ on the metabolism of serotonin (5-hydroxytryp-tamine, 5-HT) is equivocal. A number of studies consistently find a Li+-induced increase in the levels of the major metabolite, 5-hydroxyin-doleacetic acid (5-HIAA), in rat brain and in human CSF [155], which appears to reflect an increase in the rate of synthesis of 5-HT [156]. Li+-induced increases in the level of the amino acid precursor, tryptophan, and in the uptake of tryptophan by brain have also been reported [157], implying elevated tryptophan availability during Li+ treatment. In rat brain, chronic Li+ decreases the activity of tryptophan hydroxylase, the enzyme which, when activated by a Ca2+ and calmodulin-dependent protein kinase, leads to the synthesis of 5-HT [158]. Ca2+ increases the strength of binding of tryptophan to the enzyme, whereas Li+ has the opposite effect [159]. Tryptophan uptake is coupled to 5-HT utilization by a negative feedback mechanism and, therefore, the Li+-induced inhibition of tryptophan hydroxylase with a resultant decrease in 5-HT utilization could produce the observed increase in tryptophan uptake. 

Phosphoryl group transfer reactions add or remove phosphoryl groups to or from cellular metabolites and macromolecules, and play a major role in biochemistry. Phosphoryl transfer is the most common enzymatic function coded by the yeast genome and, in addition to its importance in intermediary metabolism (see Chapter 5), the reaction is catalysed by a large number of central regulatory enzymes that are often part of signalling cascades, such as protein kinases, protein phosphatases, ATPases and GTPases. 

The switch in the action of the enzyme between its kinase and phosphatase activities is brought about by phosphorylation mediated by the serine/threonine protein kinase A (PKA), the same cAMP dependent enzyme which plays a role in the control of glycogen metabolism. In its kinase form, PFK-2 is dephosphorylated but phosphorylated in the phosphatase form. 

Figure 21.9 The mitogen-activated protein kinase cascade (MAP kinase cascade). The active protein Ras activates Raf by promoting its recruitment to a cell membrane. Through a series of phosphorylations MAP kinase is activated as follows MAP kinase kinase kinase (Raf) phosphorylates MAP kinase kinase which, in turn, phosphorylates MAP kinase, the final target enzyme. MAP kinase phosphorylates transcription factors for genes that express proteins involved in proliferation. Another nomenclature for the enzymes is also used raf is MEKK MAPKK is MEK and finally ERK is MAP kinase (ERK is the abbreviation for extracellular-signal-related kinase) For comparison, the reader is referred to the metabolic phosphorylase cascade, which is discussed in Chapter 12 (Figure 12.12).

A wide variety of other biochemical effects has been reported to be associated with treatment of cells with vinblastine, vincristine, and related compounds (S). These effects include inhibition of the biosynthesis of proteins and nucleic acids and of aspects of lipid metabolism it is not clear whether such effects contribute to the therapeutic or toxic actions of vincristine and vinblastine. Vinblastine and vincristine inhibit protein kinase C, an enzyme system that modulates cell growth and differentiation (9). The pharmacological significance of such inhibition has not been established, however, and it must be emphasized that the concentrations of the drugs required to inhibit protein kinase C are several orders of magnitude higher than those required to alter tubulin polymerization phenomena (10). 

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