Signal Transduction
Somatostatin, a tetradecapeptide, is best known as an inhibitor of growth hormone secretion, but it appears to inhibit the release
of other of pituitary hormones and also insulin from pancreatic - cells. In view of this it has been suggested that its name should be changed to panhibin.
Palindrome: a string or phrase that reads the same backwards and forwards. In the present case it refers to ACTGCAGT with its complementary
sequence TGACGTCA. For more examples, see Table 19.1, page 579.
The concentration of PKA in mammalian cells is so high, in the range
0.2 – 2 mol L 1,10 that one might expect that little or no cAMP would ever be free in the cytosol and accessible to the phosphodiesterases which catalyse its hydrolysis. However, following the dissociation of the inactive R2C2 complexes, the cooperative nature of the binding of cAMP to the regulatory subunits
is lost, and with it the high-affinity state.9 As a result, the cAMP dissociates and becomes subject to conversion to 5 -AMP by phosphodiesterase (Figure 9.3). Furthermore, the activity of this enzyme may be enhanced through phosphorylation by PKA, so ensuring abrupt termination of the signal.11, 12
The specific actions of protein kinase A and other phosphorylating enzymes are controlled by targeting of the regulatory subunits. Specific anchoring proteins are present in particular locations such as the Golgi membranes, centrioles (microtubule organizing centres), cytoskeleton, etc.6, 9, 13 With four isoforms of the regulatory subunits that can form heterodimers (as in R R ), and three isoforms of the catalytic subunit, it is possible that up to 24 different forms of the holoenzyme exist, although not all isoforms are expressed in all tissues.
Protein kinase A and the regulation of transcription
Activation of the CREB transcription factor
The action of PKA in the control of glycogen metabolism is an example of short-term regulation, as between one meal and the next. PKA also acts to regulate events in the longer term by switching on the transcription of specific genes. For this purpose, the signals have to be conveyed into the
nucleus. The first identified example of such long-term control by PKA was the expression of the hypothalamic peptide somatostatin. Other well-established examples are the stimulation, by catecholamines, of the synthesis of mRNA coding for the 2-adrenergic receptor and by glycoprotein hormones that generate receptors for TSH and LH. The phosphorylation substrate is the transcription factor cAMP response element binding protein (CREB, see Figure 9.4). Following phosphorylation by PKA, the CREB dimer interacts with DNA at the cAMP response element, CRE. This is an 8 base pair palindromic sequence (TGACGTCA) generally located within 100 nucleotides of the TATA box (see page 287).
However, transcriptional partners or coactivators are necessary for subsequent activation of transcription. Such factors are CREB binding protein, CBP, and p300, both large proteins that only interact with the phosphorylated form of CREB and direct the transcription factors to the transcriptional machinery situated at the TATA box.14
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Phosphorylation and Dephosphorylation: Protein Kinases A and C
FIG 9.4 Pathways leading to activation of CREB-mediated gene expression.
The figure illustrates the basic cytosolic and nuclear events leading to the activation of the cAMP response elements (CRE). Both PKA and (Ca2 activated) CaM kinase can diffuse
into the nucleus and phosphorylate CREB at the same serine residue. The phosphorylation promotes CREB dimer formation and in this form the active transcription factor binds specifically at the CRE resulting in gene transcription. Dephosphorylation of CREB leads to attenuation of the response.
Not surprisingly, the activation of CREB can also occur as a consequence of those hormonal stimuli that cause an elevation in the cytosol concentration of Ca2 acting through AC1 (Ca2 -sensitive adenylyl cyclase 1).15 Less expected was the finding that in some cells, Ca2 can activate CREB directly as a result of phosphorylation by CaM kinases at the same residue (S133).16, 17 These effects of Ca2 may have important consequences in the medium and long term, in cells which also react abruptly to transient elevations in cytosol
Ca2 . Clearly, for cells that must be able to secrete proteins repeatedly and ‘on demand’, such as pancreatic endocrine (islet) and exocrine (acinar) cells, it is essential to restore the complement of secretory proteins after the event. The Ca2 that provides the stimulus to secretion may thus also provide the stimulus to specific protein synthesis. In pancreatic cells, Ca2 entering through voltage-sensitive channels acts both as the stimulus to the secretion of glucagon and also, through the action of CaM kinase II, as a stimulus to transcription of the glucagon gene.18 The CREB is also understood to regulate many of the long-term effects of stimulus-induced plasticity at synapses and to underlie the process of long-term potentiation.17, 19
In addition to phosphorylation, the effect of CREB on cellular responses may be also be regulated through the expression of CREB itself. In testicular Sertoli cells, follicle stimulating hormone (FSH) acting through cAMP not
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