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Fig. 2.3.2 PI3K, phosphatidylinositol-3-kinase; PLC, phospholipase C. ® represents a phosphate group (-PO42-).

Enzymes

This box briefly describes some of the enzymes common to many of the control processes discussed in this book.

Adenylyl cyclase (or adenylate cyclase): This is an integral protein of the plasma membrane. It is activated or inhibited by interaction with membrane-associated G-proteins. Adenylyl cyclase catalyses the production of cAMP from ATP (see Fig. 2.3.1).

Phospholipase C: This is also a cell membrane-bound enzyme, activated by another class of G-proteins, Gq. It catalyses the cleavage of PIP2 to release IP3 and diacylglycerol (see Fig. 2.3.2).

Phosphatidylinositol-3-kinase (PI3K, see Fig. 2.3.2) is activated by docking with protein targets of insulin receptor phosphorylation known as insulin receptor substrates (IRSs). It phosphorylates the 3'-position on the inositol ring of PIP2, forming phosphatidyl-inositol (3',4',5') trisphosphate (PIP3).

Protein kinases

There is a family of protein kinases, of which the following are particularly relevant to this discussion. The following are serine or threonine kinases, involved in regulation of enzyme activity.

cAMP-dependent protein kinase (protein kinase A, or PKA) was the first protein kinase to be identified. It is involved in rapid regulation of many pathways of energy metabolism. In its inactive state it is composed of four subunits, two regulatory (R) subunits and two catalytic (C). When cAMP binds to the R

subunits, they dissociate, leaving the catalytic subunits active against protein targets.

Protein ki nase B (PKB, also known as Akt) was first cloned as a homologue of PKA; because its properties were somewhere between those of PKA and PKC (see below) it was termed PKB!

Protein kinase C (PKC): There is a large family of PKCs, divided into four subgroups. One subgroup, the 'classical' PKCs, are activated by calcium ions (hence the name PKC). Other subgroups, the 'atypical PKCs', are activated by various lipid mediators that are generated in the cell membrane in response to other enzyme activities. These activators include diacylglycerol and PI P3.

AMP-activated protein kinase (AMP-PK): This member of the protein kinase family is activated by AMP; this activation is antagonised by ATP. Therefore AMP-PK 'senses' the cell's energy status: when there is a drain on ATP, the AMP/ATP ratio rises and AMP-PK is activated, leading in turn to inhibition of ATP-utilising pathways (particularly biosynthetic pathways) and increased ATP generation. This enzyme has therefore been described as a 'cellular fuel gauge'.

Glycogen synthase kinase 3 (GS3K): As its name suggests, GS3K was first identified as a kinase responsible for phosphorylation, and inactivation, of glycogen synthase. It is now recognised to play a role in several signal chains relevant to energy metabolism although it has retained its original name (see Box 2.4, Fig. 2.4.1).

Mitogen-activated protein kinase (MAPK) is part of a signal chain that links cell-surface receptors, especially those for growth factors including insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), with altered gene transcription in the nucleus, altered post-translational processing of proteins and control of the cell cycle. At one time the MAPK pathway was thought to be involved in insulin regulation of glycogen synthase, but it is now known that this is not so: it may be involved, however, in the regulation of gene expression by insulin. The pathway is complex, with a MAP-kinase-kinase and also a MAP-kinase-kinase-kinase!

3 -Phosphoinositide dependent kinase-1 (PDK1) is a serine/threonine kinase expressed in many tissues. It binds phosphatidylinositides that are phos-phorylated in the 3' position, particularly phosphatidylinositol (3',4',5')- tris-phosphate (PIP3 on Fig. 2.4.1, Box 2.4). This activates it, and it phosphorylates and activates (amongst other proteins) PKB.

Protein phosphatases

There is a large family of protein phosphatases involved in dephosphorylation of serine, threonine and tyrosine residues and hence regulation of enzyme activity.

Protein phosphatase-1 (PP-1) is a serine phosphatase that plays a particular role in energy metabolism. PP-1 may have a subunit that associates it with glycogen, the glycogen targeting subunit. PP-1 that is associated with glycogen is known as PP-1G. There are glycogen targeting subunits that are specific to liver and muscle. The activity of PP-1 is itself regulated: for instance, it is activated by insulin, leading to dephosphorylation and hence inactivation of glycogen synthase. (For many dephosphorylation reactions, however, it seems that the phosphatase activity is constitutively expressed (i.e. always present) and not regulated. An example is the suppression of hormone-sensitive lipase (HSL) activity in the adipocyte by insulin. Insulin reduces phosphorylation of HSL by reducing cAMP concentration and hence PKA activity; the enzyme is dephosphorylated (and inactivated) by protein phosphatases that are always active.)

Protein-tyrosine phosphatases are also important in metabolic regulation. One particular isoform, PTP1B, is responsible for dephosphorylation of tyrosine residues in the insulin receptor, and therefore turning off insulin action. PTP1B is itself regulated by phosphorylation. Insulin, via the insulin receptor tyrosine kinase, phosphorylates tyrosine residues in PTP1B and reduces its activity. cAMP leads, presumably via PKA, to serine phosphorylation of PTP1B and an increase in activity. Signalling from catecholamines can then be seen to reduce insulin signalling. PTP1B has been described as a 'critical point for insulin and catecholamine counter-regulation'.

the events involved by looking at some well-established signal chains. The signal chains drawn out as examples in Box 2.4 make an important point. One molecule of enzyme can bring about the transformation of many molecules of its substrate. Therefore signal chains such as these open up the possibility of 'amplification' of a hormonal signal within a cell, with each successive step involving larger and larger numbers of molecules. This is also often described as a 'cascade' of events following hormone-receptor binding.

Many events in signal transduction are mediated by phosphorylation, in which serine, threonine or tyrosine residues in proteins are phosphorylated (using ATP) or dephosphorylated by specific enzymes (kinases and phosphatases respectively). In general, tyrosine phosphorylation is involved in receptor function and early in signal chains. When enzymes are regulated by phosphorylation, it mostly involves serine residues. Sometimes the enzymatic activity is intrinsic to a protein with another function: for instance, the insulin receptor has tyrosine kinase activity which is activated when insulin binds. Phosphorylation or dephosphorylation of a protein leads to a conformational change, which may alter the protein's catalytic activity, or may lead to interaction with other proteins (sometimes called 'docking'). Both the kinases and the

Box 2.4 Signal transduction chains: some examples

Effects on metabolism*- PKB-P (active)-«-PKB

Effects on metabolism, x.

gene expression and mRNA translation

GS3K-P (inactive>

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