Major pathways of Intracellular Cell Signalling

Upon ligand binding, most cell surface receptors stimulate intracellular target enzymes to transmit and amplify a signal.

An amplified signal can be propagated to the nucleus to regulate gene expression in response to an external cell stimulus.

The major intracellular signalling pathways indlude the cAMP and cGMP pathways, the phospholipase C-Ca2+ pathway, the NF-kB (for nuclear factor involved in the transcription of the k light chain gene in b lymphocytes) transcription factor pathway, the Ca2+ – calmodulin pathway, the MAP (for mitogen-activated protein) kinase pathway, and the JAK-STAT (for signal transducers and activators of transcription) pathway.

1. The cAMP pathway

  • The intracellular signalling pathway mediated by cAMP was discovered in 1958 by Earl Sutherland while studying the action of epinephrine, a hormone that breaks down glycogen into glucose before muscle contraction.
  • When epinephrine binds to its receptor, there is an increase in the intracellular concentration of cAMP. cAMP is formed from adenosine triphosphate (ATP) by the action of the enzyme adenylyl cyclase and degraded to adenosine monophosphate (AMP) by the enzyme cAMP phosphodiesterase.
  • This mechanism led to the concept of a first messenger mediating a cell-signalling effect by a second messenger, cAMP. The epincphrine receptor is linked to adenylyl cyclase by G protein, which stimulates cyclase ativity upon epinephrine binding.
  • The intracellular signalling effects of cAMP are mediated by the enzyme cAMP- dependent protein kinase (or protein kinase A).
  • In its inactive form, protein kinase A is a tetramer composed of two regulatory subunits (to which cAMP-binds) and two catalytic subunits. Binding of cAMP results in the dissociation of the catalytic subunits.
  • Free catalytic subunits can phosphorylate serine residues on target proteins.
  • In the epinephrine-dependent regulation of glycogen metabolism, protein kinase A phosphorylates two enzymes:
  1. Phosphorylase kinase, which in turn phosphorylates glycogen phosphorylase to break down glycogen into glucose-I-phosphate.
  2. Glycogen synthase, which is involved in the synthesis of glycogen. Phosphorylatioon of glycogen synthase prevents the synthesis of glycogen

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2. The cGMP pathway

  • cGMP is also a second messenger. It is produced from guanosine triphosphate (GTP) by guanylate cyclase and degraded to GMP by a phosphodiesterase. Guanylate cyclases are activated by nitric oxide and peptide signalling molecules.
  • The best characterized role of cGMP is in photoreceptor rod cells of the retina, where is converts light signals to nerve impulses.

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3. Phospholipase C-Ca2+ pathway

  • Another second messenger involved in intracellular signalling derives from the phospholipid phosphatidylinositol 4.5-bisphosphate (PIP2 ) present in the inner leaflet of the plasma membrane
  • The hydrolysis of PIP2 by the enzyme phospholipase C (PLC)- stimulated by a number of hormones and growth factors-produced two second messengers: diacylglycerol and inositol 1,4,5- trisphosphate (IP3 ).
  • These two messengers stimulate two downstream signalling pathway cascades: protein kinase C and Ca2+ mobilization.
  • Two forms of PLC exist: PLC-ß and PLC-ɣ. PLC-ß is activated by G protein. PLC-Y contains SH2 domains that enable association with receptor protein tyrosine kinases.
  • Tyrosine phosphorylation increases PLC-ɣ activity, which in turn stimulates the breakdown of PIP2.
  • Diacylglycerol, derived from PIP2 hydorlysis, activates members of the protein kinase C family (protein serine and threonene kinases).
  • Phorbol esters are tumor growth- promoting agents acting, like diacylglycerol, by stimulation of protein kinase C activities. Protein kinase C activates other intracellular targets such as protein kinases of the MAP 93 kinase pathway to produce the phosphorylation of transcription factors leading to changes in gene expression and cell proliferation.

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4. NF-kB transcription factor pathway

  • NF-kB is a transcription factor involved in immune responses in several cells and is stimulated by protein kinase C In its inactive state, the NF-kB and the complex is retained in the cytoplasm.
  • The phosphorylation of I-kB –triggered by I-kB kinase—leads to the destruction of I-kB by the 26S proteasome and the release of NF-kB. The free NF-kB heterodimer translocates into the nucleus to activate gene transcription in response to immunologic and inflammatory signalling.

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5. Ca2+- calmodulin pathway

  • Although the second messenger diacylglycerlol remains associated with the plasma membrane, the other second messenger IP3, derived from PIP2, is released into the cytosol to activate ion pumps and free Ca2+ from intracellular storage sites.
  • High cytosolic Ca2+ concentrations (from a basal level of 0.1 UM to an increased 1.0 uM concentration after cytosolic release) activate several Ca2+ dependent protein kinases and phosphatases.
  • Calmodulin is a Ca2+ – dependent protein that is activated when the Ca2+ concentration increases to 0.5 uM. Ca2+ – calmodulin complexes bind to a number of cytosolic target proteins to regulate cell responses.
  • Note that Ca2+ is an important second messenger and that its intracellular concentration can be increased not only by its release from intracellular storage sites but also by increasing the entry of Ca2+ into the cell from the extracellular space.

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6. MAP kinase pathway

  • This pathway involves evolutionarily conserved protein kinases (yeast to humans) with roles in cell growth and differentiation.
  • MAP kinases are protein serine and threonine kinases activated by growth factors and other signalling molecules.
  • A well- characterized form of MAP kinase is the ERK family.
  • Members of the ERK (for extracellular signal- regulated kinase) family act through either protein tyrosine kinase or G protein—associated receptors.
  • Both cAMP and Ca2+ – dependent pathways can stimulate or inhibit the ERK pathway in different cell types.
  • The activation of ERK is mediated by two protein kinases: Raf, a protein serine or threonine kinase, which, in turn, activates a second kinase called MEK (for MAP kinase or ERK kinase).
  • Stimulation of a growth factor receptor leads to the activation of the GTPbinding protein Ras (for rat sarcoma virus), which interacts with Raf.
  • Raf phosphorylates and activates MEK, which then activates ERK by phosphorylation of serine and threonine residues.
  • ERK then phosphorylates nuclear and cytosolic target proteins.
  • In the nucleus, activated ERK phosphorylates the transcription factors EIK-I (for E-26-like protein I) and serum response factor (SRF) which recognize the regulatory sequence called serum response element (SRE).
  • In addition to ERK, mammalian cells contain two other MAfP Kinases called JNK and p38 MAP kinases.
  • Cytokines and ultraviolet irradiation stimulate JNK and p38 MAP kinase activation mediated by small GTPbinding proteins different from Ras.These kinases are not activated by MEK but by a distinct dual kinase called MKK (for MAP kinase Kinase).
  • A key element in the ERK pathway are the Ras proteins, a group of oncogenic proteins of tumor viruses that cause sarcomas in rats.
  • Mutations in the Ras gene have been linked to human cancer.
  • Ras proteins are guanine nucleotide-binding protein with functional properties similar to the G protein A subunits (activated by GTP and inactivated by guanosine diphosphate[GDP]).
  • A difference with G protein is that Ras proteins do not associate with by subunits.
  • Ras is activated by guanine nucleotide exchange factors to facilitate the release of GDP in exchange for GTP.
  • The Activity of the Ras GTP complex is terminated by GTP hydrolysis, which is stimulated by GTPase-activating proteins.
  • In human cancers, mutation f Ras genes results in a breakdown failure of GTP and , therefore, the mutated Ras protein remains continuously in the active GRP-bound form.

7. JAK-STAT pathway

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  • The preceding MAP kinase pathway links the cell surface to the nucleus signalling mediated by a protein kinase cascade leading to the phosphorylation of transcription factors.
  • The JAK-STAT pathway provides a close connection between protein tyrosine kinases and transcription factors by directly affecting transcription factors STAT (for signal transducers and activators of transcription) proteins are transcription factors with an SH2 domain and are present in the cytoplasm in an inactive state.
  • Stimulation of a receptor by ligand binding recruits STAT proteins, which bind to the cytoplasmic protion of receptorassociated JAK protein tyrosine kinase through their SH2 domain and become phosphorylated.
  • Phosphorylated STAT proteins then dimerize and translocate into the nucleus, where they activate the transcription of target genes.

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