Question

1. Outline a plausible signal transduction cascade involving PKA that upregulates transcription of a CRE-regulated gene.

2. What is the role of SH-2 domains in receptor enzyme signaling?

Answer 1

1. CREB-TF (CREB, cAMP response element-binding protein) is a cellular transcription factor. It binds to certain DNA sequences called cAMP response elements (CRE), thereby increasing or decreasing the transcription of the genes. CREB proteins are activated by phosphorylation from various kinases, including PKA, and Ca2+/calmodulin-dependent protein kinases on the Serine 133 residue. When activated, CREB protein recruits other transcriptional coactivators to bind to CRE promoter 5’ upstream region. Hydrophobic leucine amino acids are located along the inner edge of the alpha helix. These leucine residues tightly bind to leucine residues of another CREB protein forming a dimer. This chain of leucine residues forms the leucine zipper motif. The protein also has a magnesium ion that facilitates binding to DNA. Protein kinase A (PKA[N 1]) is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase.

The activation of cAMP/PKA signaling starts from ligand-dependent activation of the G protein-coupled receptors (GPCRs) and is followed by Gsα activation, adenylyl cyclase (AC) activation, and cAMP generation. cAMP activates PKA by binding to PKA regulatory subunits (RIs or RIIs). The PKA catalytic subunits (C) are then released from the PKA tetramer to phosphorylate targets in the cytoplasm, in the plasma membrane (CaV, KATP, etc.), and in the nucleus (CREB). The subcellular localization of PKA is dependent on binding of R subunits to different A-kinase anchoring proteins (AKAPs). The termination of cAMP signaling is dependent on phosphodiesterases (PDEs)-mediated degradation of cAMP to AMP.

2. The function of SH2 domains is to specifically recognize the phosphorylated state of tyrosine residues, thereby allowing SH2 domain-containing proteins to localize to tyrosine-phosphorylated sites. This process constitutes the fundamental event of signal transduction through a membrane, in which a signal in the extracellular compartment is "sensed" by a receptor and is converted in the intracellular compartment to a different chemical form, i.e. that of a phosphorylated tyrosine. Tyrosine phosphorylation leads to activation of a cascade of protein-protein interactions whereby SH2 domain-containing proteins are recruited to tyrosine-phosphorylated sites. This process initiates a series of events which eventually result in altered patterns of gene expression or other cellular responses. The SH2 domain, which was first identified in the oncoproteins Src and Fps, is about 100 amino-acid residues long. It functions as a regulatory module of intracellular signaling cascades by interacting with high affinity to phosphotyrosine-containing target peptides in a sequence-specific and strictly phosphorylation-dependent manner.