Cellular substrates, phosphorylation

Phosphorylation of cellular substrates by PKA occurs on serine and threonine residues. 

Due to the problems in identification of cellular substrates of protein kinases, as described in Chapter 7, it has been a difficult and lengthy process to determine the functionally relevant substrates. Pig. 13.11 gives an overview of the ceU-cycle-specific activation of CDKs and some important substrates. Comparatively sparse information is available on the Gj and S phase substrates of the CDKs. In contrast, many proteins have been described that undergo specific phosphorylation in G2/M phase. The sequence (K/R)-S/T-P-X-K (X any amino acid) has been identified as a consensus sequence for phosphorylation by CDKs. 

The biochemical mechanisms by which cAMP-dependent phosphorylation leads to enhanced IR-PTH release remain to be determined. It is of interest, however, that isoproterenol activates phosphorylation of proteins of similar molecular weight in the rat parotid gland (22), while glucagon stimulates phosphorylation of a protein of molecular weight 19,000 in calcitonin-secreting cultured cells from a medullary carcinoma of the rat thyroid (22) It is conceivable that in all three tissues, activation of exocytosis results from a cAMP-dependent phosphorylation of a critical cellular substrate. 

N is often limiting in the marine environment. Further, many enzymes are sensitive to cellular substrate concentrations rather than extracellular concentrations and it is difficult to measure the relevant intracellular metabohte pools. In vitro assays may affect the conformation of enzymes and the degree to which they are modified. For example, allosteric effects (see Section 1.3.3) may be modified under in vitro conditions. Many enzymes undergo posttranslational regulation wherein enzyme activity is affected by binding of activator/inactivator proteins and covalent modification of the enzyme (e.g., adenylylation, phosphorylation or carbamylation) (Ottaway, 1988). When there is posttranslational modification of enzymes, enzyme activity measured in assays may be unrelated to in vivo activity (see Section 2.2.1) and there are few ways to determine the extent of enzyme modification in nature. 

Vanadate (VOj or H2VO4 ) was first recognized in 1979 as having insulin mimetic properties [258]. Since then, vanadate and vanadyl (V ) have been shown to mimic most but not all biological actions of insulin in vitro and to lower blood glucose in streptozotocin-treated rats [259, 260]. Vanadate is a potent inhibitor of phosphotyrosine phosphatases, an interesting activity since the insulin receptor is a tyrosine kinase, and some of the actions of insulin have been proposed to take place via autophosphorylation of the insulin receptor and phosphorylation of cellular substrates on tyrosine residues [261]. Some recent developments on the mechanism and the in vivo activity of vanadate and its derivatives are presented here. 

The first evidence of a cellular substrate of the insulin receptor kinase came from White et al. (1985b) who described a 185 kDa phosphoprotein in Fao hepatoma cells which was rapidly phosphorylated upon insulin stimulation. ppl85, recently renamed IRS-1, is a cytosolic protein with at least 30 potential serine/threonine- and 10 potential tyrosine-phosphorylation sites (Sun et al., 1991). Six of these tyrosine-phosphorylation sites lie in the amino acid sequence motif Tyr-Met-Xaa-Met which is recognized in its phosphorylated form by the SH2 (src homology 2) domain of the phosphatidylinositol 3-kinase (PI 3-kinase) (Sun et al., 1991). 

For HSV at least three mechanisms have been described that generate resistance to AC V deficiency or loss of viral TK activity, alteration in substrate specificity of the virus-encoded TK, and alteration in the substrate specificity of the viral DNA polymerase (1,8). Most of the ACVr mutants that have been isolated in vitro and recovered from clinical specimens are TK-deficient (TK). However, resistant clinical mutants that have an altered TK or altered DNA polymerase activity have occasionally been described too. Although TK mutants are crossresistant with drugs that also depend on viral TK for their activation (i.e., GCV, penciclovir and brivudin (BVDU), they remain sensitive to agents, such as PFA, vidarabine (Ara-A), and the acyclic nucleoside phosphonate (ANP) analogs. PFA, a pyrophosphate analog, is a direct inhibitor of the viral DNA poly-merase in which it binds to the site involved in releasing the pyrophosphate product of DNA synthesis. Phosphorylation of Ara-A to Ara-A triphosphate is carried out by cellular enzymes phosphorylation of ANP derivatives to their mono- and diphosphoryl derivatives is also carried out by cellular enzymes. 

The exact mechanism of IFN-a activity in CML is unknown, but is complex with multiple effects on cellular function. Some of the proposed alterations include changes in gene transcription, substrate phosphorylation, antigen presentation, and apoptosis. ... 

Trophic factors exemplified by NGF and its family members, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) all utilize increased tyrosine phosphorylation of cellular substrates to mediate neuronal cell survival. Actions of the NGF family of neurotrophins are not only dictated by ras activation through the Trk family of receptor tyrosine kinases, but also a survival pathway defined by phosphatidylinositol-3-kinase activity (Yao and Cooper, 1995), which gives rise to phosphoinositide intermediates that activate the serine/ threonine kinase Akt/PKB (Dudek et al., 1997). Induction of the serine-threonine kinase activity is critical for cell survival, as well as cell proliferation. Hence, for many trophic factors, multiple proteins constitute a functional multisubunit receptor complex that activates rai-depen-dent and ras-independent intracellular signaling. 

Biosynthesis of ATP. ATP is the irrunediate product of all cellular processes leading to the chemical storage of energy. It is biosynthesized by phosphorylation of ADP in the course of Substrate phosphorylation (see). Oxidative phosphorylation (see) and non-cyclic Photophosphorylation (see) in plants. Energy in the form of a third phosphate may also be transferred to ADP from other high-energy phosphates, such as creatine phosphate (see Creatine) or other nucleoside triphosphates, or in the adenylate kinase reaction. 

Antibody-mediated crosslinking of some cell surface GPI-proteins in lymphoid cells can cause phosphorylation of cellular substrates, increases in intracytoplasmic cal-... 

From the above discussion it will be seen that some 40 P (36 by oxidative and 4 by substrate phosphorylation), each with an energy content of 8-5 kcal/g-mol, are synthesised per g-mol of glucose respired and some 340 kcal therefore conserved as utilisable energy. Since the overall decrease in free energy involved in the complete oxidation of a g-mol of glucose is 686 kcal (eqn. 14, p. 85) this means that approximately 50% of this energy is trapped during respiration in a form which can be used in cellular metabolism for the synthesis of cell constituents or to perform work. 

The dose-dependent dual effects of staurosporine characterize it as a functional partial agonist of NGF (Rasouly et al., 1992). Staurosporine does not activate NGF recep-tor-trk tyrosine kinase activity (figure 3A) (Rasouly and Lazarovici, 1994) or downstream trk cellular substrates such as erk s or SNT proteins (Rasouly et al., 1996). However, staurosporine was found to induce the tyrosine-phosphorylation of a 145 kD protein (figure 3B), related to the neurotropic effects, with an unknown physiological action (Rasouly and Lazarovici, 1994). 

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