Cytosolic protein tyrosine

As work with vanadium compounds and diabetes in cell system has continued, it has become clear that there are also insulin-independent mechanisms at work. One insulin-independent signal transduction pathway appears to be involved in glycogen metabolism reactions in rat adipocytes [137] that also involve PI-3K. A major difference was that only vanadate promoted glycogenesis through the activation of a cytosolic protein tyrosine kinase, which was mediated in an insulin receptor-independent manner. 

Vanadate stimulates protein kinases in the cytosol, as demonstrated in adipose cells and extracts. The activation of a membrane and cytosolic protein tyrosine kinase have been demonstrated in adipocytes, and the membranous enzyme has been postulated to be a way to involve PI-3K actions without activation of insulin receptor substrate-1 (IRS-1) in the insulin signal transduction pathway [140], It is always difficult to determine if protein kinase activation is direct or the result of stimulation of a protein phosphatase. The fact that kinase stimulation was seen in isolated extracts after cell disintegration in this adipocyte cell system supports the idea that vanadium addition to cells could directly stimulate kinases via an as-yet-undetermined mechanism. In other experiments with 3T3-L1 adipocytes bis(acetylacetonato)oxovana-dium (IV) BMOV and bis(l-N-oxide-pyridine-2thiolato)oxovanadium (TV) caused increased tyrosine phosphorylation of both the insulin receptor and IRS-1 in a synergistic way with insulin, as measured by antibodies to phosphotyrosine residues [141]. 

Carton AJ, Flint AJ, Tonks NK (1996) Identification of pl30(cas) as a substrate for the cytosolic protein tyrosine phosphatase PTP-PEST. Mol Cell Biol 16 6408-6418... 

Signal transduction Hydrolysis of activating phosphotyrosine residue on cytosolic protein tyrosine kinases... 

Signal transduction (1) Activation of cytosolic protein tyrosine kinases (2) PI-3 kinase pathway (3) IP3/DAG pathway (4) Ras-MAP kinase pathway... 

We turn now to a second important class of cell-surface receptors, the cytokine receptors, whose cytosolic domains are closely associated with a member of a family of cytosolic protein tyrosine kinases, the JAK kinases. A third class of receptors, the receptor tyrosine kinases (RTKs), contain intrinsic protein tyrosine kinase activity in their cytosolic domains. The mechanisms by which cytokine receptors and receptor tyrosine kinases become activated by ligands are very similar, and there is considerable overlap in the intracellular signal-transduction pathways triggered by activation of receptors in both classes. In this section, we first describe some similarities in signaling from these two receptor classes. We then discuss the JAK-STAT pathway, which is initiated mainly by activation of cytokine receptors. 

Figure 1 Insulin signal transduction cascade (simplified). Intracellular kinases affected by tyrosine phosphorylation activation/deactivation under phosphotyrosine phosphatase (PTPase) regulation (vanadium-inhibitable) include (especially) IRS-I, IRS-2, she, and MAPK. V indicates possible sites of vanadium s mechanism of action. Cytosolic protein tyrosine kinase (CytPTK, not shown) stimulation by phosphatase inhibition is independent of the insulin cascade, but is also multi-step, and is particularly susceptible to vanadyl stimulation...

Aoki N, Matsuda T A cytosolic protein-tyrosine phosphatase PTPIB specifically dephos-phorylates and deactivates prolactin-activated STATSa and STATSb. J Biol Chem 2000 275 39718-39726. 

In spite of having no intrinsic catalytic domains, activation of T lymphocytes commences with tyrosine phosphorylations, activation of PLC-v with production of IP3 and DAG, and elevation of cytosolic free Ca2+. Thus, the consequences of receptor ligation are not dissimilar from those induced by the receptors for EGF or PDGF. An early study trying to explain the induction of tyrosine kinase activity resulted in the discovery of the nonreceptor protein tyrosine kinase Lck (p56lck), a T-cell-specific member of the Src family. Lck is associated with the cytosolic tail of CD4 (in helper T cells) or CD8 (in cytotoxic T cells) (Figure 8.14). As mentioned, the extracellular domains of these... 

Nonreceptor protein tyrosine kinases contain a catalytic domain, as well as various regulatory domains important for proper functioning of the enzyme. NRPTKs are found in the inner leaflet of the plasma membrane, cytosol, endosomal membranes and nucleus. These include the Src, Jak, Abl, Tec, Ack, Csk, Fak, Fes, Frk and Syk subfamilies (Fig. 24-3). Since a great deal is known about the structure and regulation of the Src family tyrosine kinase, we will use it to illustrate the principles in NRPTK signaling unique features in other subfamilies will be indicated... 

The integrins do not have any enzyme activity in their own cytoplasmic domain, but on hgand binding, stimulation of tyrosine phosphorylation is observed on the cytoplasmic side of many cells, such as fibroblasts and platelets. The exact configuration of protein-protein interactions on the cytosolic side of the integrins is not clear and the mechanism of stimulation of protein tyrosine kinases is unknown. Some components of the focal adhesion points, such as the structural protein tensin, have SH2 and SH3 domains that may serve as specific attachment points for tyrosine kinases and other signal proteins. 

Exercise was found to cause a 50% increase in o-tyrosine, m-tyrosine, and dityrosine in mitochondrial proteins but not cytosolic proteins of rat heart muscle. This increase was transient, and levels returned to normal when exercised animals were allowed to rest. There was also a transient increase in the level of o,o -dityrosine in the urine of exercised rats (L8). A single bout of exhaustive running or endurance training for 12 weeks significantly increased the level of protein carbonyls in rat skeletal muscles (R8, W14). Extensive running of rats induces a 40% increase in protein carbonyls in the lung (Rl). 

The best-studied protein tyrosine phosphatases are the high molecular weight cytoplasmic enzymes of the FTP family. X-ray structures of the human FTP IB cytosolic tyrosine phosphatase, have been solved by David Barford et and that of a Yersinia tyrosine phosphatase by Fauman et In Fig. 3.9a and b the structures of the... 

Many members of this class of receptors have an enzymatic activity known as a protein tyrosine kinase within their cytoplasmic segment. This kinase phosphorylates tyrosine residues in the receptors themselves (autophosphory lation), and in other proteins to initiate biochemical cascades. Phosphorylatipn of tyrosine can be reversed by protein tyrosine phosphatases, which are also present in all cells (Shenolikar and Naim, 1990). Tyrosine phosphatases form a diverse family of proteins, some of which are cytosolic while others are transmembrane molecules analogous to receptors. Some members of the transmembrane class may be involved in the mechanism of bacterial and viral infections (Tonks, 1991). Thus, kinases and phosphatases together act as on-off switches in the a ctivation of receptors and other proteins. 

It has recently been shown that receptors without intrinsic tyrosine kinase activity in their cytoplasmic region may nevertheless activate tyrosine kinases upon ligand binding. Phosphorylation by these kinases may directly influence events such as transcription. Thus, the binding of interferon to its receptor, which lacks an intrinsic tyrosine kinase, results in the phosphorylation of three cytosolic proteins on tyrosines. These factors associate with each other, move to the nucleus, and activate the transcription of genes that are induced by interferon treatment (Marx, 1992a). 

Not so much is known about the significance of protein tyrosine phosphatases (PTP). hihibition of PTPs (by vanadate) leads to increase in tyrosine phosphorylation, secretion and aggregation (biazuer ail, 1990 Pumigliae/a/., 1992). At least two PTPs appear to be involved in platelet aggregatioa PTPIB is proteolytically cleaved and activated by calpain and released into the cytosol during aggregation. SH-PTPl associates with the... 

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). 

Receptor tyrosine kinases (RTKs) are activated (phosphorylated) by inhibition of a negatively regulating phosphatase upon treatment with UV (A, B, or C), hydrogen peroxide, or iodoacetamide. The phosphatase activity, (i.e., dephosphorylation and inactivation of RTKs) is restored upon the addition of thiol-regenerating agents, if not inhibited irreversibly by iodoacetamide [20]. H2O2 not only inactivates membrane-bound phosphatases but also diminishes cytosolic general protein tyrosine phosphatase activity in mouse fibroblasts [21]. Further, the activation of JNK by sodium arsenite, which is reactive towards thiols (especially vicinal dithiols), is by inactivation of a JNK phosphatase [22]. 

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