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14.2Basic Mechanisms of Intracellular Signal Transduction

This section takes a closer look at the mechanisms of signal transduction. Specifically, it outlines the common mechanisms and molecules involved in intracellular signal transduction to help readers understand what exactly is meant by the “transduction” of signals or “activation.” These basic factors receive signals from upstream molecules and turn ON (activated), then they pass on the signals downstream and turn OFF again (inactivated). Readers are advised to clarify between the activated form and inactivated form of each factor in different cases.

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14.2.1

Phosphorylation and dephosphorylation of proteins

The most important mechanism in intracellular signal transduction is the phosphorylation of the amino acids tyrosine, serine, and threonine. Phosphorylation is the chemical modification process in which amino acids such as tyrosine receives a phosphate group from adenosine-5′-triphosphate (ATP) (see Chapter 4) (Figure 14-2). The enzymes catalyzing phosphorylation are known as protein kinases, or sometimes simply referred to as kinases. In most proteins that undergo phosphorylation, activation is equivalent to phosphorylation except in some cases. For instance, the activation of a specific receptor protein by a signal molecule is essentially equivalent to the phosphorylation of tyrosine, serine, or threonine among the intracellular amino acids that make up the receptor protein.

However, phosphorylation is a reversible reaction. The process by which enzymes work to reverse phosphorylation by hydrolyzing phosphates bound by phosphorylation is called dephosphorylation. The enzymes involved in the process are called phosphatases. Dephosphorylated proteins are inactivated. The phosphorylation and dephosphorylation processes are regulated by various mechanisms discussed in this chapter and in Chapter 15.

A brief explanation is provided here by taking mitogen-activated protein (MAP) kinases as an example. MAP kinases are known to transmit signals downstream through a series of phosphorylation reactions. Ras, a small G protein (described later), is activated by signals transduced from receptors. Activated Ras activates the kinase pathway to phosphorylate MAP kinase. In turn, phosphorylated MAP kinases phosphorylate various target proteins (Figure 14-3). This chain reaction of phosphorylation is called the kinase cascade, which functions to amplify small signals.

Fig. 14-2 Activation by phosphorylation of protein

A) Phosphorylation of serine, threonine, and tyrosine. B) Which amino acid to phosphorylate in the target protein is specific to the type of the kinase. The figure shows tyrosine kinase as an example.

Fig. 14-3 MAP kinase cascade

Ras activates the Raf kinase (MAP kinase kinase kinase: kinase of kinase of MAP kinase) and phosphorylates (activates) the MAP kinase ERK kinase (MEK: MAP kinase kinase). The activated MEK phosphorylates the MAP kinase (ERK). The activated ERK phosphorylates various target proteins.

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14.2.2

G1 phase

G proteins are a family of intracellular proteins that bind to a guanosine triphosphate (GTP) (see Selection 5 of Chapter 6, Figure 6-11) and convert (or hydrolyze) GTP to guanosine diphosphate (GDP) under certain conditions. G proteins are inactive when bound to GDP and active when bound to GTP. They play a crucial role in signal transduction, along with phosphorylation and dephosphorylation.

G proteins can be divided into 2 groups. One consists of small G proteins with molecular weights between 20,000 and 30,000 that function as monomers. As shown in Figure 14-4A, small G proteins usually exist in the inactive GDP-bound form. However, they are activated by an activator called guanine nucleotide exchange factor (GEF), which replaces GDP with GTP after receiving signals from receptors. Once the small G proteins complete their role, GTP is hydrolyzed to GDP, and the small G proteins are inactivated. The other group consists of trimeric G proteins made up of 3 subunits: α, β, and γ, as illustrated in Figure 14-4B. A trimeric G protein binds to a G protein-coupled receptor, a characteristic receptor that penetrates the membrane 7 times (see Chapter 15). GDP binds to the α subunit, thereby inactivating the trimeric G protein. When a signal molecule binds to the receptor, the receptor itself functions as an activator like GEF to free the GDP from the α subunit, to which GTP binds instead, thereby activating the G protein. Figure 14-4B describes the series of actions taking adenylate cyclase (see the later section on cyclic AMP) as an example of the target protein. The activated α subunit undergoes structural changes and binds with inactive adenylate cyclase to activate it. The activated adenylate cyclase then changes ATP to cyclic AMP (discussed later).

Once the α subunit completes its role, it detaches from the adenylate cyclase and hydrolyzes the bound GTP to GDP to become an inactivated G protein. G proteins have numerous types of subunits. Receptors that receive signals and target substances to act on differ by each type of subunit. G proteins have a function to hydrolyze GTP to GDP (i.e., GTPase activity). The protein known to promote this function is the GTPase activating protein (GAP) (see Figure 14-4A). In this way, GEF functions to activate G proteins, whereas GAP functions to inactivate them, mutually carrying out regulation in opposite directions.

Figure 14-4 G protein cycle

A) The G protein switches between the GTP-bound activated form and GDP-bound inactivated form. GEF dissociates GDP from the inactivated G protein and substitutes it with the GTP in the cytoplasm. The GTP in the activated G protein becomes GDP when GTPase is activated by GAP, thereby inactivating the G protein. B) Activation of adenylate cyclase by trimeric G proteinf

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Small G proteins

This group of G proteins includes approximately 200 amino acids. Many were identified from the DNA sequence as being genes similar to Ras. When their functions were analyzed later, it was found that G protein molecules have similar domains (G1–G5), which form a GTP-binding pocket when the sequence is folded (the G1–G5 domains also exist in the α subunit of trimeric G proteins.) All small G proteins switch between the GTP-bound and GDP-bound forms. Three-dimensional structural analysis has been completed for some small G proteins and the mechanism underlying their interaction with downstream molecules in the signal pathways is also being investigated. Mutations in the form of activated Ras, which induces transformation, are often seen at residue positions 12 (G1) and 61 (G3), which are important amino acids in GTP binding. Other than Ras, small G proteins also have subgroups that regulate not only cell proliferation signals but also various intracellular functions.

Some examples of small G proteins are those belonging to the Rho family; these include Rho, Rac, and Cdc42, which regulate the dynamic movement of cytoskeletal filaments such as actin. Other examples include Rab, which is involved in intracellular transport; Arf, which is involved in the formation of cellular vesicles; and Ran, which determines the directionality of the nuclear–cytoplasmic transport.

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14.2.3

Low-molecular-weight second messengers

Fig 14.5 Activation of A-kinase by cAMP

A-kinase is inactive as a heterotetramer. However, when cAMP binds to the regulatory subunit, the catalytic subunit dissociates, allowing A-kinase to demonstrate catalytic activity. When cAMP dissociates, A-kinase becomes a tetramer again and hence inactivated.

In intracellular signal transduction, not only proteins but also low-molecular substances such as cyclic adenosine 3′,5′-monophosphate (AMP), Ca2+, and inositol trisphosphate (IP3) play important roles as second messengers. These low-molecular-weight messengers transmit information to spatially distant areas by intracellular diffusion and spreading. They also amplify the stimuli of the first messengers many-fold and transduce them within the cell.

■Cyclic AMP
Cyclic AMP (cAMP) was the first second messenger discovered. ATP, which is present throughout the cytoplasm, is converted to cAMP by the activated enzyme adenylate cyclase (see Figure 14-4B). When cAMP completes its mission it is inactivated by cyclic AMP phosphodiesterase. The cellular concentration of cAMP can be boosted 20-fold within seconds after receiving stimulation from the extracellular environment. It is therefore able to support prompt and precise reactions to low-concentration signal molecules such as hormones. cAMP is also promptly broken down.

A-kinase is one of the target proteins that are activated by cAMP (Figure 14-5). An inactive A-kinase forms a tetramer with catalytic and activity-regulatory subunits. When the intracellular concentration of cAMP increases, cAMP binds to the regulatory subunit; this separates regulatory subunits from catalytic subunits, allowing A-kinase to demonstrate catalytic activity and phosphorylate the target protein. Subsequently, cAMP dissociates from the regulatory subunit, the regulatory and catalytic subunits bind together again, and the A-kinase becomes inactivated. The dissociated cAMP is promptly converted to AMP by cyclic AMP phosphodiesterase in the cytoplasm.

■Calcium ions
Calcium ions (Ca2+; see Column Selection 1 Chapter 15) also play an important role as second messengers in signal transduction. Ca2+ are maintained at a very low concentration within the cytoplasm (1/10000 of its extracellular concentration), owing to the mechanism that actively releases Ca2+ within the cytoplasm to the extracellular environment through the cell membrane (active transport) and the mechanism for active transport and storage of Ca2+ into the endoplasmic reticulum within the cell (Figure 14-6A). Some amount of calcium exists within the cytoplasm not in an ionic state but instead bound to proteins.

Fig 14.6 Signal transduction by Ca2+

A) Main mechanisms maintaining Ca2+ concentration in the cytoplasm at low levels. B) Increase in intracellular Ca2+ levels. Ca2+ waves formed at the moment of fertilization are shown by adding Ca2+ sensitive fluorescent dyes to cells. C) Cytoplasm Ca2+ concentration is low, whereas the concentration is higher in the endoplasmic reticulum; when the endoplasmic reticulum is stimulated, the Ca2+ channel in the endoplasmic reticulum opens, releasing large amounts of Ca2+ to the cytoplasm. D) Activation of calmodulin by Ca2+

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Inositol phospholipid

Other than the ones mentioned in the text, there are many substances such as cyclic GMP and inositol working as second messengers within the cell.
As described in Chapter 11, the cell membrane is mainly composed of phospholipids. Of these phospholipids, phosphatidylinositol 4,5-bisphosphate (PIP2) is broken down into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) by the enzyme phospholipase C (PLC), which is activated when cell surface receptors receive extracellular signals (Column Figure 14-1). The small molecule IP3 moves away from the cell membrane and spreads in the cell to function as a second messenger, as shown in the example in Figure 14-6C. Ca2+ released from signals transduced by inositol phospholipid is also a second messenger. Both are hence molecules taking part in signal transduction within the cells, triggered by signals of first messengers from outside the cell.

Column Figure 14-1 Production of IP3 and DAG from PIP2

PIP2 of the cell membrane is broken down into IP3 and DAG by the activation of PLC. DAG remains in the cell membrane, whereas IP3 is dispersed inside the cytoplasm.

In the cytoplasm, the Ca2+ concentration rises immediately after a signal which uses Ca2+ as a second messenger is received. The reception of the messenger opens the Ca2+ channels (see Chapter 11) in the cell membrane or endoplasmic reticulum, causing a Ca2+ rush into the cytoplasm owing to the high Ca2+ concentration gradient (which had been maintained until the opening of the channel). Figure 14-6B shows images of a fertilized egg immediately after fertilization, with Ca2+ in the cell visualized for observation. Figure 14-6C shows an example of the process by which the Ca2+ stored within the intracellular endoplasmic reticulum is mobilized into the cytoplasm (see Column).

Some of the proteins in the cytoplasm require Ca2+ to function. For instance, the protein calmodulin changes its three-dimensional structure after being bound to Ca2+ to form an activated calcium-calmodulin complex. Some protein kinases are calmodulin-dependent kinases that demonstrate kinase activity only when the activated calcium-calmodulin complex is bound to them (Figure 14-6D).

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14.2.4

Signal transduction by proteolysis/span>

All intracellular signal transduction mechanisms discussed in the previous paragraphs are reversible. However, some receptors on the cell membrane or proteins within the cytoplasm undergo cleavage or degradation as a means of signal transduction. Once the protein undergoes cleavage or degradation, it will not return to its original state. Some examples of such signal transduction mechanisms are those seen with receptors involved in differentiation and caspases involved in apoptosis*2(see Column Selection 3 Chapter 14).

*2 In Greek, “apo” means “from” and “ptosis” means “falling.” Defoliation is also phenomena to which apoptosis contributes.

Figure 14-7 shows a specific example. Caspases are proteolytic enzymes that have important roles in apoptosis and are usually present in the cytoplasm as inactive precursors. When apoptotic signals are transduced to cells, the inhibition of the pathways that activate the caspases is canceled. Caspase precursors begin by mutually degrading themselves with the aid of activation factors, to eventually become activated caspases. Then, the cascade of proteolytic reactions is initiated by the caspases to complete apoptosis.

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