Mechanisms of Signal Transduction
Signal transduction at the cellular level refers to the movement of signals from outside the cell to inside. The movement of signals can be simple, like that associated with receptor molecules of the acetylcholine class: receptors that constitute channels which, upon ligand interaction, allow signals to be passed in the form of small ion movement, either into or out of the cell. These ion movements result in changes in the electrical potential of the cells that, in turn, propagates the signal along the cell. More complex signal transduction involves the coupling of ligand-receptor interactions to many intracellular events. These events include phosphorylations by tyrosine kinases and/or serine/threonine kinases. Protein phosphorylations change enzyme activities and protein conformations. The eventual outcome is an alteration in cellular activity and changes in the program of genes expressed within the responding cells.
Please refer to the page on Growth Factors for descriptions of the growth factors described in this page and the explanation of their abbreviations.
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Classifications of Signal Transducing Receptors
Signal transducing receptors are of three general classes:
- 1. Receptors that penetrate the plasma membrane and have intrinsic enzymatic activity Receptors that have intrinsic enzymatic activities include those that are tyrosine kinases (e.g. PDGF, insulin, EGF and FGF receptors), tyrosine phosphatases (e.g. CD45 [cluster determinant-45] protein of T cells and macrophages), guanylate cyclases (e.g. natriuretic peptide receptors) and serine/threonine kinases (e.g. activin and TGF-b receptors). Receptors with intrinsic tyrosine kinase activity are capable of autophosphorylation as well as phosphorylation of other substrates.
- Additionally, several families of receptors lack intrinsic enzyme activity, yet are coupled to intracellular tyrosine kinases by direct protein-protein interactions (see below).
- 2. Receptors that are coupled, inside the cell, to GTP-binding and hydrolyzing proteins (termed G-proteins). Receptors of the class that interact with G-proteins all have a structure that is characterized by 7 transmembrane spanning domains. These receptors are termed serpentine receptors. Examples of this class are the adrenergic receptors, odorant receptors, and certain hormone receptors (e.g. glucagon, angiotensin, vasopressin and bradykinin).
- 3. Receptors that are found intracellularly and upon ligand binding migrate to the nucleus where the ligand-receptor complex directly affects gene transcription.
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Receptor Tyrosine Kinases (RTKs)
The proteins encoding RTKs contain four major domains:
- An extracellular ligand binding domain.
- An intracellular tyrosine kinase domain.
- An intracellular regulatory domain.
- A transmembrane domain.
The amino acid sequences of the tyrosine kinase domains of RTKs are highly conserved with those of cAMP-dependent protein kinase (PKA) within the ATP binding and substrate binding regions. Some RTKs have an insertion of non-kinase domain amino acids into the kinase domain termed the kinase insert. RTK proteins are classified into families based upon structural features in their extracellular portions (as well as the presence or absence of a kinase insert) which include the cysteine rich domains, immunoglobulin-like domains, leucine-rich domains, Kringle domains, cadherin domains, fibronectin type III repeats, discoidin I-like domains, acidic domains, and EGF-like domains. Based upon the presence of these various extracellular domains the RTKs have been sub-divided into at least 14 different families.
Characteristics of the Common Classes of RTKs
| Class | Examples | Structural Features of Class |
| I | EGF receptor, NEU/HER2, HER3 | cysteine-rich sequences |
| II | insulin receptor, IGF-1 receptor | cysteine-rich sequences; characterized by disulfide-linked heterotetramers |
| III | PDGF receptors, c-Kit | contain 5 immunoglobulin-like domains; contain the kinase insert |
| IV | FGF receptors | contain 3 immunoglobulin-like domains as well as the kinase insert; acidic domain |
| V | vascular endothelial cell growth factor (VEGF) receptor | contain 7 immunoglobulin-like domains as well as the kinase insert domain |
| VI | hepatocyte growth factor (HGF) and scatter factor (SC) receptors | heterodimeric like the class II receptors except that one of the two protein subunits is completely extracellular. The HGF receptor is a proto-oncogene that was originally identified as the Met oncogene |
| VII | neurotrophin receptor family (trkA, trkB, trkC) and NGF receptor | contain no or few cysteine-rich domains; NGFR has leucine rich domain |
Many receptors that have intrinsic tyrosine kinase activity as well as the tyrosine kinases that are associated with cell surface receptors contain tyrosines residues, that upon phosphorylation, interact with other proteins of the signaling cascade. These other proteins contain a domain of amino acid sequences that are homologous to a domain first identified in the c-Src proto-oncogene (c - designates the cellular form of proto-onogenes that were first identified in transforming retrovirus). These domains are termed SH2 domains (Src homology domain 2). Another conserved protein-protein interaction domain identified in many signal transduction proteins is related to a third domain in c-Src identified as the SH3 domain.
The interactions of SH2 domain containing proteins with RTKs or receptor associated tyrosine kinases leads to tyrosine phosphorylation of the SH2 containing proteins. The result of the phosphorylation of SH2 containing proteins that have enzymatic activity is an alteration (either positively or negatively) in that activity. Several SH2 containing proteins that have intrinsic enzymatic activity include phospholipase C-g (PLC-g), the proto-oncogene c-Ras associated GTPase activating protein (rasGAP), phosphatidylinositol-3-kinase (PI-3K), protein phosphatase-1C (PTP1C), as well as members of the Src family of protein tyrosine kinases (PTKs).
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Non-Receptor Protein Tyrosine Kinases (PTKs)
There are numerous intracellular PTKs that are responsible for phosphorylating a variety of intracellular proteins on tyrosine residues following activation of cellular growth and proliferation signals. There is now recognized two distinct families of non-receptor PTKs. The archetypal PTK family is related to the Src protein. The Src protein is a tyrosine kinase first identified as the transforming protein in Rous sarcoma virus. Subsequently, a cellular homolog was identifed as c-Src. Numerous proto-oncogenes were identified as the transforming proteins carried by retroviruses. The second family is related to the Janus kinase (Jak).
Most of the proteins of both families of non-receptor PTKs couple to cellular receptors that lack enzymatic activity themselves. This class of receptors includes all of the cytokine receptors (eg the interleukin-2 (IL-2) receptor) as well as the CD4 and CD8 cell surface glycoproteins of T cells and the T cell antigen receptor (TCR). This mode of coupling receptors to intracellular PTKs suggests a split form of RTK.
Another example of receptor-signaling through protein interaction involves the insulin receptor (IR). This receptor has intrinsic tyrosine kinase activity but does not directly interact, following autophosphorylation, with enzymatically active proteins containing SH2 domains (e.g. PI-3K or PLC-g). Instead, the principal IR substrate is a protein termed IRS-1. IRS-1 contains several motifs that resemble SH2 binding consensus sites for the catalytically active subunit of PI-3K. These domains allow complexes to form between IRS-1 and PI-3K. This model suggests that IRS-1 acts as a docking or adapter protein to couple the IR to SH2 containing signaling proteins.
Additional adapter proteins have been identified, the most commonly occurring being a protein termed growth factor receptor-binding protein 2, Grb2.
An example of an alteration in receptor activity in response to association with an intracellular PTK is the nicotinic acetylcholine receptor (AChR). These receptors comprise an ion channel consisting of four distinct subunits (a, b, g, and d). The b, g, and d subunits are tyrosine phosphorylated in response to acetylcholine binding which leads to an increase in the rate of desensitization to acetylcholine.
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Receptor Serine/Threonine Kinases (RSTKs)
The receptors for the TGF-b superfamily of ligands have intrinsic serine/threonine kinase activity. There are more than 30 multifunctional proteins of the TGF-b superfamily which also includes the activins, inhibins and the bone morphogenetic proteins (BMPs). This superfamily of proteins can induce and/or inhibit cellular proliferation or differentiation and regulate migration and adhesion of various cell types. The signaling pathways utilized by the TGF-b, activin and BMP receptors are different than those for receptors with intrinsic tyrosine kinase activity or that associate with intracellular tyrosine kinases.
At least 17 RSTKs have been isolated and can be divided into 2 subfamilies identified as the type I and type II receptors. Ligands first bind to the type II receptors which then leads to interaction with the type I receptors. When the complex betwen ligand and the 2 receptor subtypes forms, the type II receptor phosphorylates the type I receptor leading to initiation of the signaling cascade. One predominant effect of TGF-b is regulation of progression through the cell cycle. One nuclear protein involved in the responses of cells to TGF-b is the proto-oncogene, c-Myc ("mick") which directly affects the expression of genes harboring Myc-binding elements.
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Non-Receptor Serine/Threonine Kinases
The are several serine/threonine kinases that function in signal transduction pathways. The two more commonly known are cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). Additional serine/threonine kinases important for signal transduction are the mitogen activated protein kinases (MAP kinases).
Protein Kinase C (PKC)
PKC was originally identified as a serine/threonine kinase that was maximally active in the presence of diacylglycerols (DAG) and calcium ion. It is now known that there are at least ten proteins of the PKC family. Each of these enzymes exhibits specific patterns of tissue expression and activation by lipid and calcium. PKCs are involved in the signal transduction pathways initiated by certain hormones, growth factors and neurotransmitters. The phosphorylation of various proteins, by PKC, can lead to either increased or decreased activity. Of particular importance is the phosphorylation of the EGF receptor by PKC which down-regulates the tyrosine kinase activity of the receptor. This effectively limits the length of the cellular responses initiated through the EGF receptor.
MAP Kinases
MAP kinases were identified by virtue of their activation in response to growth factor stimulation of cells in culture, hence the name mitogen activated protein kinases. MAP kinases are also called ERKs for extracellular-signal regulated kinases. On the basis of in vitro substrates the MAP kinases have been variously called microtubule associated protein-2 kinase (MAP-2 kinase), myelin basic protein kinase (MBP kinase), ribosomal S6 protein kinase (RSK-kinase: i.e. a kinase that phosphorylates a kinase) and EGF receptor threonine kinase (ERT kinase). All of these proteins have similar biochemical properties, immuno-crossreactivities, amino acid sequence and ability to in vitro phosphorylate similar substrates.
Maximal MAP kinase activity requires that both tyrosine and threonine residues are phosphorylated. This indicates that MAP kinases act as switch kinases that transmits information of increased intracellular tyrosine phosphorylation to that of serine/threonine phosphorylation. Although MAP kinase activation was first observed in response to activation of the EGF, PDGF, NGF and insulin receptors, other cellular stimuli such as T cell activation (which signals through the Lck [lick] tyrosine kinase), phorbol esters (that function through activation of PKC), thrombin, bombesin and bradykinin (that function through G-proteins) as well as N-methyl-D-aspartate (NMDA) receptor activation and electrical stimulation rapidly induce tyrosine phosphorylation of MAP kinases.
MAP kinases are, however, not the direct substrates for RTKs nor receptor associated tyrosine kinases but are in fact activated by an additional class of kinases termed MAP kinase kinases (MAPK kinases) and MAPK kinase kinases (MAPKK kinases). One of the MAPK kinases has been identified as the proto-oncogenic serine/threonine kinase, Raf. Ultimate targets of the MAP kinases are several transcriptional regulators e.g. serum response factor (SRF), and the proto-oncogenes Fos, Myc and Jun as well as members of the steroid/thyroid hormone receptor super family of proteins.
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Phospholipases and Phospholipids in Signal Transduction
Phospholipases and phospholipids are involved in the processes of transmitting ligand-receptor induced signals from the plasma membrane to intracellular proteins. The primary protein affected by the activation of phospholipases is PKC which is maximally active in the presence of calcium ion and DAG. The generation of DAG occurs in response to agonist activation of various phospholipases. The principal mediators of PKC activity are receptors coupled to activation of phospholipase C-g (PLC-g). PLC-g contains SH2 domains that allow it to interact with tyrosine phosphorylated RTKs. This allows PLC-g to be intimately associated with the signal transduction complexes of the membrane as well as membrane phospholipids that are its substrates. Activation of PLC-g leads primarily to the hydrolysis of membrane phosphatidylinositol bisphosphate (PIP2) leading to an increase in intracellular DAG and inositol trisphosphate (IP3). The released IP3 interacts with intracellular membrane receptors leading to an increased release of stored calcium ions. Together, the increased DAG and intracellular free calcium ion concentrations lead to increased activity of PKC.
Recent evidence indicates that phospholipases D and A2 (PLD and PLA2) also are involved in the sustained activation of PKC through their hydrolysis of membrane phosphatidylcholine (PC). PLD action on PC leads to the release of phosphatidic acid which in turn is converted to DAG by a specific phosphatidic acid phosphomonoesterase. PLA2 hydrolyzes PC to yield free fatty acids and lysoPC both of which have been shown to potentiate the DAG mediated activation of PKC. Of medical significance is the ability of phorbol ester tumor promoters to activate PKC directly. This leads to elevated and unregulated activation of PKC and the consequent disruption in normal cellular growth and proliferation control leading ultimately to neoplasia.
Phosphatidylinositol-3-Kinase (PI-3K)
PI-3K is tyrosine phosphorylated, and subsequently activated, by various RTKs and receptor-associated PTKs. PI-3K is a heterodimeric protein containing an 85 kDa and 110 kDa subunits. The p85 subunit contains SH2 domains that interact with activated receptors or other receptor-associated PTKs and is itself subsequently tyrosine phosphorylated and activated. The 85 kDa subunit is non-catalytic, however, it does contain a domain homologous to GTPase activating (GAP) proteins. It is the 110 kDa subunit that is enzymatically active. PI-3K, associates with and is activated by, the PDGF, EGF, insulin, IGF-1, HGF and NGF receptors. PI-3K phosphorylates various phosphatidylinositols at the 3 position of the inositol ring. This activity generates additional substrates for PLC-g allowing a cascade of DAG and IP3 to be generated by a single activated RTK or other protein tyrosine kinases.
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G-Protein Coupled Receptors
There are several different classifications of receptors that couple signal transduction to G-proteins. These classes of receptor are termed G-protein coupled receptors, GPCRs. Well over 1000 different GPCRs have been cloned, most being orphan receptors having no as yet identified ligand. Three different classes of GPCR are reviewed:
- 1. GPCRs that modulate adenylate cyclase activity. One class of adenylate cyclase modulating receptors activate the enzyme leading to the production of cAMP as the second messenger. Receptors of this class include the b-adrenergic, glucagon and odorant molecule receptors. Increases in the production of cAMP leads to an increase in the activity of PKA in the case of b-adrenergic and glucagon receptors. In the case of odorant molecule receptors the increase in cAMP leads to the activation of ion channels. In contrast to increased adenylate cyclase activity, the a-type adrenergic receptors are coupled to inhibitory G-proteins that repress adenylate cyclase activity upon receptor activation.
- 2. GPCRs that activate PLC-g leading to hydrolysis of polyphosphoinositides (e.g. PIP2) generating the second messengers, diacylglycerol (DAG) and inositoltrisphosphate (IP3). This class of receptors includes the angiotensin, bradykinin and vasopressin receptors.
- 3. A novel class of GPCRs are the photoreceptors. This class is coupled to a G-protein termed transducin that activates a phosphodiesterase which leads to a decrease in the level of cGMP. The drop in cGMP then results in the closing of a Na+/Ca2+ channel leading to hyperpolarization of the cell. See the Role of Vitamin A in Vision for more details.
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G-Protein Regulators
The activity of G-proteins with respect to GTP hydrolysis is regulated by a family of proteins termed GTPase activating proteins, GAPs. The proto-oncogenic protein, Ras, is a G-protein involved in the genesis of numerous forms of cancer (when the protein sustains specific mutations). Of particular clinical significance is the fact that oncogenic activation of Ras occurs with higher frequency than any other gene in the development of colo-rectal cancers. Regulation of Ras GTPase activity is controlled by rasGAP.
There are several other GAP proteins besides rasGAP that are important in signal transduction. There are two clinically important proteins of the GAP family of proteins. One is the gene product of the neurofibromatosis type-1 (NF1) susceptibility locus. The NF1 gene is a tumor suppressor gene and the protein encoded is called neurofibromin. The second is the protein encoded by the BCR locus (break point cluster region gene). The BCR locus is rearranged in the Philadelphia+ chromosome (Ph+) observed with high frequency in chronic myelogenous leukemias (CMLs) and acute lymphocytic leukemias (ALLs).
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Intracellular Hormone Receptors
Hormone receptors are proteins that effectively bypass all of the signal transduction pathways described thus far by residing within the cytoplasm. Additionally, all of the hormone receptors are bi-functional. They are capable of binding hormone as well as directly activating gene transcription.
The steroid/thyroid hormone receptor superfamily (e.g. glucocorticoid, vitamin D, retinoic acid and thyroid hormone receptors) is a class of proteins that reside in the cytoplasm and bind the lipophilic steroid/thyroid hormones. These hormones are capable of freely penetrating the hydrophobic plasma membrane. Upon binding ligand the hormone-receptor complex translocates to the nucleus and bind to specific DNA sequences termed hormone response elements (HREs). The binding of the complex to an HRE results in altered transcription rates of the associated gene.
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Phosphatases in Signal Transduction
Substantial evidence links both tyrosine and serine/threonine phosphorylation with increased cellular growth, proliferation and differentiation. Removal of the incorporated phosphates must be a necessary event in order to turn off the proliferative signals. This suggests that phosphatases may function as anti-oncogenes or growth suppressor genes. The loss of a functional phosphatase involved in regulating growth promoting signals could lead to neoplasia. However, examples are known where dephosphorylation is required for promotion of cell growth. This is particularly true of specialized kinases that are directly involved in regulating cell cycle progression. Therefore, it is difficult to envision all phosphatases as being tumor suppressor genes.
There are two broad classes of protein tyrosine phosphatases (PTPs). One class are transmembrane enzymes which contain the phosphatase activity domain in the intracellular portion of the protein. The other are intracellularly localized enzymes. The first transmembrane PTP characterized was the leukocyte common antigen protein, CD45. This protein was shown to have homology to the intracellular PTP, PTP1B. There are at least six sub-classes of the transmembrane PTPs.
The clearest studies of a role for transmembrane PTPs in signal transduction have involved the CD45 protein. These studies have shown that CD45 is involved in the regulation of the tyrosine kinase activity of lck in T cells. As indicated above Lck is associated with T cell antigens CD4 and CD8 generating a split-RTK involved in T cell activation. It is suspected that CD45 dephosphorylates a regulatory tyrosine phosphorylation site in the C-terminus of Lck, thereby, increasing the activity of Lck towards its substrate(s).
The second class of PTPs are the intracellular proteins. The C-terminal residues of most if not all intracellular PTPs are very hydrophobic and suggest these sites are membrane attachment domains of these proteins. One role of intracellular PTPs is in the maturation of Xenopus oocytes in response to hormone. Over expression of PTP-1B in oocytes resulted in a marked retardation in the rate of insulin- and progesterone-induced maturation. These results suggest a role for PTP-1B in countering the signals leading to cellular activation.
The above observation as well as several others have demonstrated a link between insulin function and PTP-1B. PTP-1B directly interacts with the insulin receptor and removes the tyrosine phosphates incorporated by autophosphorylation in response to insulin binding, thereby, negatively affecting the activity of the insulin receptor. Recently the PTP-1B gene was disrupted in mice by targeted deletion. Mice lacking a functional PTP-1B gene exhibit increased insulin sensitivity as well as resistance to obesity induced by a high fat diet.
As with the transmembrane PTPs little is known about the regulation of the activity of the intracellular PTPs.
Two intracellular PTPs (PTP-1C and PTP-1D) have been shown to contain SH2 domains. These SH2 domains allow these PTPs to directly interact with tyrosine phosphorylated RTKs and PTKs, thereby, dephosphorylating tyrosines in these proteins. Following receptor stimulation of signal transduction events, the SH2 containing PTPs are directed to several of the RTKs and/or PTKs with the net effect being a termination of the signaling events by tyrosine dephosphorylation
Other phosphatases that recognize serine and/or threonine phosphorylated proteins also exist in cells. These are referred to as protein serine phosphatases (PSPs). At least 15 distinct PSPs have been identified. The type 2A PSPs exhibit selective substrate specificity towards PKC phosphorylated proteins; in particular serine and threonine phosphorylated receptors. Type 2A PSPs are more effective than other PSPs in dephosphorylating RSKs, proteins that are involved in signaling cascades by phosphorylating ribosomal S6 protein (see above). However, a type 1 PSP is required to dephosphorylate S6 itself.
The type 2A PSPs have 2 subunits (a regulatory and a catalytic) both of which can associate with one of the tumor antigens of the DNA tumor virus, polyoma. Transformation by DNA tumor viruses such as polyoma appears to be mediated by the formation of a signal transduction unit consisting of a virally encoded T antigen and several host encoded proteins. Several host proteins are tyrosine kinases of the src family. Polyoma middle T antigen also can bind to PI-3K. The association of type 2A PSPs in these complexes may lead to dephosphorylation of regulatory serine/threonine phosphorylated sites resulting in increased signal transduction and subsequent cellular proliferation.
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This article has been modified by Dr. M. Javed Abbas.
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22:51 19/12/2002
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