Direct modulation of the protein kinase a catalytic subunit α by growth factor receptor tyrosine kinases

Protein kinases are enzymes that transmit signals throughout the intracellular environment. They catalyze the transfer of a phosphate group from ATP to serine, threonine and tyrosine residues within downstream substrates and in this manner alter the biochemical properties of the target proteins. Cyclic-AMP-dependent protein kinase A (PKA) is a member of the large family of AGC protein kinases that also includes PKC, PKB, Rsk and many others. It is one of the most well studied protein kinases to date with a multitude of structural and biochemical studies providing detailed information about how the enzyme functions [Taylor et al., 2008; Taylor et al., 2005]. PKA exists in cells as an inactive holoenzyme consisting of a dimer of two allosteric R (regulatory) subunits, each of which binds to a C (catalytic) subunit. The catalytic subunit (PKA-C) is composed of a conserved catalytic core (aa. 40–300) that is flanked by short amino and carboxy terminal sequences, termed N and C tails. It is now appreciated that the C-tail (residues 301–350) contains particular regions that function as cis-acting regulatory components of PKA catalytic activity [Kannan et al., 2007; Taylor et al., 2008]. An acidic cluster of amino acids (residues 327–336, FDDYEEEEIR), termed the active site tether (AST) is highly dynamic and regulates ATP binding, organization of the active site and the recognition and recruitment of peptide and protein substrates [Batkin et al., 2000; Chestukhin et al., 1996; Kannan et al., 2007; Kennedy et al., 2009; Taylor et al., 2008; Yang et al., 2009]. In cells, the catalytic subunit is assembled as a fully phosphorylated and active enzyme that is kept dormant by its association with an R subunit, in a holoenzyme complex. PKA is typically activated in response to extracellular cues that induce the production of the second messenger, cyclic adenosine monophosphate (cAMP). cAMP binds to the R subunits which induces a dramatic conformational change and the subsequent release of the active C subunits. PKA-C is then free to phosphorylate serine and theronine residues on numerous intracellular substrates that can be found in the cytosol, cytoskeleton, plasma membrane and nucleus. The discrete subcellular compartmentalization of PKA by the A kinase anchoring proteins (AKAPs) provides an important degree of specificity to PKA signaling by helping to match a given extracellular stimulus to a specific subset of cellular targets [Scott, 2003]. PKA-mediated signal transduction controls a myriad of cellular processes including gene transcription, proliferation, differentiation, migration and survival. Growth factors are one type of extracellular cue known to activate PKA [Bornfeldt and Krebs, 1999; Ciardiello and Tortora, 1998; Fishman et al., 1997]. Soluble peptide growth factors such as epidermal growth factor (EGF) and platelet derived growth factor (PDGF), initiate their effects upon binding to their cognate receptor tyrosine kinases (RTKs), the EGF receptor (EGFR) and PDGF receptor (PDGFR), respectively. Once active, these receptors initiate a host of downstream signaling pathways (eg. AKT/PKB, PKC and Erk) which promote gene transcription, cell proliferation, survival and migration [Hubbard and Miller, 2007]. There are numerous reports that demonstrate a role for PKA in signalling events that occur downstream of the activated EGFR and PDGFRs. A prime example is the demonstration that the RI and C subunits of PKA physically interact with the activated EGFR in a Grb2-dependent manner [Ciardiello and Tortora, 1998]. This interaction was reportedly important for the ability of the EGFR to promote mitogenic signaling events [Ciardiello et al., 1999]. PKA can directly phosphorylate the EGFR and inhibit its tyrosine kinase activity in vitro and cAMP analogs attenuate EGF-induced tyrosine phosphorylation of the EGFR in mammalian fibroblasts [Barbier et al., 1999]. However, PKA’s effect on the EGFR may be cell type specific as PKA was shown to stimulate tyrosine phosphorylation of the EGFR resulting in enhanced kinase activity in PC12 and A431 cells [Piiper et al., 2003]. In response to stimulation of cells with PDGF, PKA is activated and translocated from the cell membrane [deBlaquiere et al., 1994; Graves et al., 1996], and it can either promote or inhibit cellular proliferation and migration depending upon the cell type studied [Bornfeldt and Krebs, 1999; Bornfeldt et al., 1995; deBlaquiere et al., 1994; Deming et al., 2008; Graves et al., 1993; Graves et al., 1996; Howe et al., 2005; Jalvy et al., 2007; O’Connor and Mercurio, 2001; Stork and Schmitt, 2002]. While these connections have been known for some time, the precise manner in which growth factor receptors and PKA activity intersect is poorly understood. The results reported here demonstrate that tyrosyl phosphorylation of PKA regulates its activity and identify a molecular mechanism for crosstalk between growth factor receptor tyrosine kinases and PKA signaling networks.