Intracellular signal transduction is a chain of The targets often...

Pathways of Intracellular Signal Transduction

Intracellular signal transduction is a chain of reactions that transmits signals from the cell surface to intracellular targets.

The targets often include transcription factors that regulate gene expression.

Intracellular signaling was first studied in hormones such as epinephrine, which signals the breakdown of glycogen to glucose.

In 1958 Sutherland discovered that the action of epinephrine was mediated by an increase in cyclic AMP (cAMP), leading to the concept of cAMP as a second messenger.

Pathways of Intracellular Signal TransductioncAMP is formed from ATP by adenylyl cyclase and degraded to AMP by

cAMP phosphodiesterase.

The epinephrine receptor is coupled to adenylyl cyclase via a G protein that stimulates enzymatic activity, increasing the concentration of cAMP.

Pathways of Intracellular Signal Transduction

Effects of cAMP in animal cells are mediated by cAMPdependent protein kinase, or protein kinase A.

The inactive form has two regulatory and two catalytic subunits. cAMP binds to the regulatory subunits, which dissociate.

The free catalytic subunits can then phosphorylate serine on target proteins.

Pathways of Intracellular Signal TransductionIn glycogen metabolism, protein kinase A phosphorylates two enzymes:

• Phosphorylase kinase is activated, and in turn activates glycogen phosphorylase.

• Glycogen synthase is inactivated.

So, glycogen breakdown is stimulated and glycogen synthesis is blocked.

Pathways of Intracellular Signal Transduction

Signal amplification: each molecule of epinephrine activates one receptor.

Each receptor may activate up to 100 molecules of Gs, which then stimulates the adenylyl cyclase, which can catalyze the synthesis of many cAMP.

Each molecule of protein kinase A phosphorylates many molecules of phosphorylase kinase, and so forth.

Pathways of Intracellular Signal Transduction

Increased cAMP can activate transcription of genes that contain a regulatory sequence—the cAMP response element, or CRE.

The free catalytic subunit of protein kinase A goes to the nucleus and phosphorylates the transcription factor CREB(CRE-binding protein).

This leads to expression of cAMP-inducible genes.

Pathways of Intracellular Signal Transduction

Protein phosphorylation is rapidly reversed by the action of protein phosphatases, which terminates responses initiated by receptor activation of protein kinases.

Pathways of Intracellular Signal Transduction

cAMP can also directly regulate ion channels:It is a second messenger in sensing smells—

odorant receptors are G protein-coupled; they stimulate adenylyl cyclase, leading to an increase in cAMP.

cAMP opens Na+ channels in the plasma membrane, leading to initiation of a nerve impulse.

Cyclic GMP (cGMP) is also an important second messenger in animal cells.

cGMP is formed from GTP by guanylyl cyclases and degraded to GMP by a phosphodiesterase.

cGMP mediates biological responses such as blood vessel dilation.

Pathways of Intracellular Signal Transduction

In the vertebrate eye, cGMP is the second messenger that converts visual signals to nerve impulses.

The photoreceptor in rod cells of the retina is a G protein-coupled receptor called rhodopsin.

Rhodopsin is activated when light is absorbed by the associated small molecule retinal.

Rhodopsin then activates the G protein transducin; the α subunit stimulates cGMP phosphodiesterase, leading to decreased levels of cGMP.

cGMP levels are translated to nerve impulses by a direct effect of cGMP on ion channels.

Figure 15.24 Role of cGMP in photoreception

Pathways of Intracellular Signal TransductionTwo major pathways of intracellular signaling use second messengers

derived from the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2).

Hydrolysis of PIP2 by phospholipase C produces two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3).

Pathways of Intracellular Signal TransductionThere are two forms of phospholipase C:

PLC-β is stimulated by G proteins.PLC-γ has SH2 domains that associate with receptor protein-tyrosine kinases.

Tyrosine phosphorylation increases PLC- γ activity, stimulating hydrolysis of PIP2.

Pathways of Intracellular Signal Transduction

DAG remains associated with the plasma membrane and activates protein-serine/threonine kinases of the protein kinase C family.

IP3 is a small polar molecule that is released to the cytosol, where it signals release of Ca2+

from the ER.

Pathways of Intracellular Signal Transduction

Cytosol concentration of Ca2+ is maintained at an extremely low level by Ca2+ pumps.

IP3 stimulates release Ca2+ from the ER by binding to receptors that are ligand-gated Ca2+

channels.

Increased Ca2+ affects activity of several proteins, including protein kinases and phosphatases. Calmodulin is activated when Ca2+

concentration increases.

Ca2+/calmodulin then binds to target proteins, including protein kinases.

Pathways of Intracellular Signal Transduction

The CaM kinase family are activated by Ca2+/calmodulin; they phosphorylate metabolic enzymes, ion channels, and transcription factors.

One form of CaM kinase regulates synthesis and release of neurotransmitters.

One transcription factor phosphorylated by CaM kinase is CREB, which is also phosphorylated by protein kinase A.

This illustrates one of many intersections between the Ca2+ and cAMP signaling pathways.

These pathways function coordinately to regulate many cellular responses.

Pathways of Intracellular Signal Transduction

Ca2+ is also increased by uptake of extracellular Ca2+ by regulated channels in the plasma membrane.

In electrically excitable cells of nerve and muscle, voltage-gated Ca2+ channels are opened by membrane depolarization.

The resulting increase in intracellular Ca2+

signals the further release of Ca2+ from the ER by opening Ca2+ channels (ryanodine receptors) in the ER membrane.

Ca2+ is a versatile second messenger that controls a wide range of cellular processes.

Figure 15.29 Regulation of intracellular Ca2+ in electrically excitable cells

Pathways of Intracellular Signal Transduction

PIP2 is also the start of another signaling pathway.

PIP2 is phosphorylated by phosphatidylinositide (PI) 3-kinase.

This yields the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3).

Pathways of Intracellular Signal TransductionPIP3 targets a protein-serine/threonine kinase called Akt and also binds

protein kinase PDK1.

Activation of Akt also requires protein kinase mTOR (in a complex called mTORC2) which is also stimulated by growth factors.

Pathways of Intracellular Signal Transduction

Akt phosphorylates several target proteins, transcription factors, and other protein kinases.

Transcription factors include members of the Forkhead or FOXO family.

Akt phosphorylation of FOXO sequesters it in inactive form.

Pathways of Intracellular Signal Transduction

If growth factors are not present, Akt is not active, and FOXO travels to the nucleus where it stimulates transcription of genes that inhibit cell proliferation, or induce cell death.

Figure 15.32 Regulation of FOXO (Part 1)

Figure 15.32 Regulation of FOXO (Part 2)

Pathways of Intracellular Signal Transduction

Protein kinase GSK-3 is also inhibited by Akt phosphorylation.

GSK-3 targets include the translation initiation factor eIF2B.

Phosphorylation of eIF2B leads to a global downregulation of translation initiation.

Pathways of Intracellular Signal Transduction

The mTOR pathway couples the control of protein synthesis to the availability of growth factors, nutrients, and energy.

The mTORC1 complex is activated downstream of Akt and functions to regulate cell size, by controlling protein synthesis.

Figure 15.33 The mTOR pathway

Pathways of Intracellular Signal Transduction

mTORC1 phosphorylates two targets that regulate protein synthesis:

S6 kinase—controls translation by phosphorylating ribosomal protein S6.

eIF4E binding protein-1 (4E-BP1)—interacts with initiation factor eIF4E, which binds to the 5′ cap of mRNAs.

Pathways of Intracellular Signal Transduction

The MAP kinase pathway is a cascade of protein kinases that is highly conserved in evolution, found in all eukaryotic cells.

MAP kinases (mitogen-activated protein kinases) are protein-serine/threonine kinases.

Pathways of Intracellular Signal Transduction

MAP kinases initially characterized in mammalian cells belong to the ERK (extracellular signal-regulated kinase) family.

Activation of ERK is mediated by two upstream protein kinases, which are coupled to growth factor receptors by the Ras GTP-binding protein.

Pathways of Intracellular Signal Transduction

Activation of Ras leads to activation of the Raf protein serine/threonine kinase, which phosphorylates and activates a second protein kinase called MEK (for MAP kinase/ERK kinase).

Figure 15.34 Activation of the ERK MAP kinases

Pathways of Intracellular Signal Transduction

Ras proteins are guanine nucleotide-binding proteins that function like αsubunits of G proteins.

Ras is activated by guanine nucleotide exchange factors that stimulate exchange of GDP for GTP.

Ras-GTP activity is terminated by GTP hydrolysis, stimulated by the interaction of Ras-GTP with GTPase-activating proteins.

Figure 15.35 Regulation of Ras proteins

Pathways of Intracellular Signal Transduction

Mutations of ras genes in human cancers inhibit GTP hydrolysis by Ras proteins.

The mutated Ras proteins therefore remain continuously in the active GTP-bound form, driving the proliferation of cancer cells even in the absence of growth factor stimulation.

Molecular Medicine 15.1 Cancer: Signal Transduction and the ras Oncogenes: A human colon polyp (an early stage of colon cancer)

Pathways of Intracellular Signal Transduction

One mode of Ras activation is mediated by receptor protein-tyrosine kinases.

Autophosphorylation of these receptors results in association with Ras guanine nucleotide exchange factors as a result of SH2-mediated protein interactions.

Figure 15.36 Ras activation downstream of receptor protein-tyrosine kinases

Pathways of Intracellular Signal Transduction

In its active GTP-bound form, Ras binds with Raf protein-serine/threonine kinase.

Raf initiates a protein kinase cascade leading to ERK activation.

A fraction of activated ERK goes to the nucleus where it regulates transcription factors by phosphorylation.

Figure 15.37 Induction of immediate-early genes by ERK

Pathways of Intracellular Signal Transduction

A primary response to growth factor stimulation is rapid transcriptional induction of immediate-early genes.

This is mediated by a regulatory sequence called the serum response element (SRE), which is recognized by a transcription factors including the serum response factor (SRF) and Elk-1.

Pathways of Intracellular Signal Transduction

Many immediate-early genes encode transcription factors, so their induction leads to altered expression of a battery of other downstream genes called secondary response genes.

Pathways of Intracellular Signal Transduction

Yeasts and mammalian cells have multiple MAP kinase pathways.

In mammalian cells, three groups of MAP kinases have been identified: the ERK family, and the JNK and p38 MAP kinases.

Figure 15.38 Pathways of MAP kinase activation in mammalian cells