Phosphorylation of substrates

Morris CM, Smith GJ (1992) Altered levels and protein kinase C-mediated phosphorylation of substrates in normal and transformed mouse lung epithelial cells. Exp CeU Res 200 149-155... 

The signaling mechanisms activated by neurotrophic factors, which include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are fundamentally different from those discussed for G protein-coupled receptors and Ca " (Russell and Duman 2002). The neurotrophic factors bind to specific receptors, TrkA, TrkB, and TrkC (the name Trk is derived from their identification as troponin/receptor kinases from colon carcinoma) (Fig. 2). The Trk receptors contain an extracellular binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. Two neurotrophic factor molecules are required for activation of a Trk receptor dimer, resulting in activation of the tyrosine kinase domains and phosphorylation of substrate proteins as well as autophosphorylation of the Trk receptor itself. 

Fig. 6.2. Regulation of protein kinase A via cAMP. Protein kinase A is a tetrameric enyzme composed of two catalytic subunits (C) and two regulatory subunits (R). In the R2C2 form, protein kinase A is inactive. Binding of cAMP to R leads to dissociation of the tetrameric enyzme into the R2 form with bound cAMP and free C subunits. In the free form, C is active and catalyzes the phosphorylation of substrate proteins (S) at Ser/Thr residues.

Fig. 6.4. Formation and function of diacylglycerol and Ins(l,4,5)P3. Formation of diacylglycerol (DAG) and Ins(l,4,5)P3 is subject to regulation by two central signaling pathways, which start from transmembrane receptors with intrinsic or associated tyrosine kinase activity (see Chapters 8 11) or from G-protein-coupled receptors. DAG activates protein kinase C (PKC, see Chapter 7), which has a regulatory effect on ceU proliferation, via phosphorylation of substrate proteins. Ins(l,4,5)P3 binds to corresponding receptors (InsPs-R) and induces release of Ca from internal stores. The membrane association of DAG, PtdIns(3,4)P2 and PL-C is not shown here, for clarity.

Fig. 8.3. Ligand-induced autophosphorylation and substrate phosphorylation of receptor tyrosine kinases. The tyrosine kinase domain of the receptor tyrosine kinase is activated by ligand binding. Consequently, autophosphorylation and/or phosphorylation of substrate proteins takes place. The substrate proteins possess specific phosphotyrosine binding domains (SH2 in the figure or FTP domains, see 8.2), which bind to phosphate residues formed in the process of autophosphorylation.

Autophosphorylation and phosphorylation of substrate proteins are essential elements of signal transduction via receptor tyrosine kinases. Autophosphorylation fre-... 

Mechanism of activation of the epidermal growth factor (EGF) receptor, a representative receptor tyrosine kinase. The receptor polypeptide has extracellular and cytoplasmic domains, depicted above and below the plasma membrane. Upon binding of EGF (circle), the receptor converts from its inactive monomeric state (/eft) to an active dimeric state (right), in which two receptor polypeptides bind noncovalently. The cytoplasmic domains become phosphorylated (P) on specific tyrosine residues (Y) and their enzymatic activities are activated, catalyzing phosphorylation of substrate proteins (S). 

As discussed above, two of the established effectors of the Ga limb of heterotrimeric G-proteins are adenylate cyclase (AC) and phospholipase C (PLCP), which, on stimulation, lead to the generation of the second-messenger molecules, cAMP and DAG/IP3, respectively. Through the respective activation of cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC), GPCRs coupled to AC and PLCp have the potential to effect indirect modulation of neurotransmitter release by phosphorylation of substrate proteins involved in the... 

Fig. 3. Artificial substrates for the insulin receptor tyrosyl kinase. Shows the Km values exhibited by basal and insulin-stimulated kinase activities together with the insulin-stimulated increase in Vmax for a variety of substrates. These include angiotensin and its modified derivative (VAL-5), also synthetic peptides of Glu Tyr and the so-called sarc -peptide, which bears the sequence around the tyrosyl autophosphorylation site of the sarc protein. Data are also given for a G-protein mixture of Gj/G0. These studies (referred to in the text) all employed soluble, purified insulin receptor preparations. No evidence has yet been presented for tyrosyl phosphorylation of substrates using isolated membrane preparations containing insulin receptors.

Figure 13.6 Insulin-receptor signalling. Insulin binding to its receptor causes the phosphorylation of substrate proteins, in this case IRS-1 IRS-1 mediates the recruitment of GLUT-4 transporters to the plasma membrane. IRS-1 also has effects on glycogen synthesis, glycolysis and fatty acid production.

The insulin receptor is the prototype of a t3rrosine kinase receptor with a constimtive, oligomeric structure (Chapter 1). The receptor has been cloned and characterized in detail, notably by Axel Ullrich, R. C. Kahn and colleagues, see Chapter 1.29 Binding of insulin stimulates the intrinsic receptor tyrosine kinase. A crystal structure of the tyrosine kinase domain of the insulin receptor was solved. This leads to phosphorylation of tyrosine residues and to the recruitment and subsequent phosphorylation of substrates. 

After insulin binding, protein phosphorylation and dephosphorylation appear to play a pivotal role in further signal transmission from the insulin receptor to the effector systems (Goldstein, 1992). Since at the post-kinase level tyrosine phosphorylation of substrate proteins is involved in insulin signalling, an important role for tyrosine phosphatases in the regulation of post-kinase signalling mechanisms has to be assumed (Goldstein, 1992). 

I 72. Which one of the following enzymes catalyzes high-energy phosphorylation of substrates during glycolysis ... 

Besides fueling biosynthetic processes, ATP hydrolysis also serves to spark many metabolic reactions. Thus, phosphorylation of substrates - sugars, for example -often initiates their metabolic transformation. In addition, a number of receptor-mediated, regulatory mechanisms depend on the splitting of ATP (or GTP). 

The common catalytic function of protein kinases is the covalent phosphorylation of substrate proteins via transfer of the y-phosphate of ATP to the OH group of serine, threonine or tyrosine residues. This catalytic function is carried out by a catalytic do-... 

Another area of concern involves the inherent nonspecificity of protein kinase-catalyzed phosphorylation in general, especially in broken-cell systems. Often, introduction of a kinase to broken-cell fractions results in the enzyme s catalyzing the phosphorylation of substrates that would not normally be phosphorylated by the kinase in the intact cell. PKG, for example, is often not highly selective in recognizing substrates in vitro and will catalyze the phosphorylation of proteins that are endogenous and physiologi-... 

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