The results presented in this report describe an unprecedented role for tyrosine phosphorylation of the C subunit of PKA during growth factor signaling and build upon the existing role of the C tail as a critical regulatory region. The C tail allosterically regulates the enzyme on two levels; it directly regulates the catalytic core and functions to facilitate interactions with cellular components that modulate catalytic activity [Kannan et al., 2007
]. Our data show that RTKs directly phosphorylate PKA-C on a tyrosine residue/s and identified Y330 within the C tail as the major phosphorylation site in vitro
. We demonstrate that phosphorylation of PKA-C by RTKs functionally modifies the enzymatic activity of PKA in vitro
and lastly establish that this phosphorylation event occurs in response to stimulation of cells with several different growth factors.
Although the functional relevance of the AST of PKA-C has been extensively studied with regard to how it allosterically regulates the catalytic core, post-translational modification of this regulatory region has never been reported. In mammalian cells, post-translational modification of PKA-C is reported to occur through autophosphorylation on serine residues (S10, S110, and S338) and phosphorylation in the activation loop on T197 by PDK1 or a PDK1-like enzyme [Cheng et al., 1998
; Yonemoto et al., 1993
]. Large scale phosphoproteomic screens performed in mouse brain and cell lines expressing oncogenic tyrosine kinases have also observed phosphorylation on tyrosine 69, however, the kinase/s responsible for this phosphorylation event and the functional relevance have not yet been determined [Ballif et al., 2008
; Guo et al., 2008
; Rikova et al., 2007
]. It is formally possible that other tyrosine residues in addition to Y330 are phosphorylated by RTKs at a lower abundance. However, given that there was no detectable tyrosine phosphorylation on sites other than Y330 via mass spectrometry and that mutation of Y330 to Y330F ablated phosphorylation, it is likely that Y330 is the major, if not only, residue phosphorylated by the PDGFR and EGFR.
The kinetic studies described here demonstrate that tyrosine phosphorylation of the catalytic subunit enhances the enzymatic activity of PKA. Y330 specifically appears to be involved in the hydrophobic packing of the backbone of the β1 strand that flanks the glycine rich loop () [Yang et al., 2009
]. During the catalytic cycle, Y330 moves into close proximity to the p-3 subsite of the substrate, a site known to confer specificity for PKA [Kemp et al., 1977
; Taylor et al., 1990
; Walsh et al., 1992
]. A previous report by Batkin et al. demonstrated that mutation of Y330 to a panel of amino acids resulted a marked increase in the Km
and decrease in catalytic efficacy of PKA-C [Batkin et al., 2000
]. Interestingly, of the mutants tested, the phenylalanine mutant (Y330F) exhibited the smallest although yet significant difference in Km
as compared to control PKA-C. As conversion of tyrosine to phenylalanine retains the phenyl ring but loses the phenolic hydroxyl these results suggested that both the phenyl ring and phenolic hydroxyl of Y330 contribute to the Km
of substrate. This is thought to occur through packing or Van der Waals interactions between the phenyl ring and residues in the conserved core and hydrogen bonding between the phenolic hydroxyl and the basic amino acid at the p-3 position, respectively. The decreased Km
observed with the tyrosine phosphorylated PKA-C subunit are consistent with this idea that Y330 is involved in creating preferential affinity of kinase for substrate as the addition of a phosphate to the hydroxyl group would be predicted to strengthen a hydrogen bonding interaction with the basic amino acid at p-3 (). The timing of Y330 phosphorylation in EGF and PDGF-treated cells was coincident with growth factor-induced PKA activity (), and the tyrosine phosphorylated C subunit was restricted to the pseudopod of chemotaxing fibroblasts, a structure previously reported to have highly localized PKA activity [Howe et al., 2005
]. However, whether phosphorylation of Y330 confers specificity for PKA to a particular subset of substrates or is required for growth factor-induced PKA kinase activity remains to be determined.
A bioinformatics study revealed that the C-tail of AGC kinases is highly conserved, suggesting a global mode of regulation for this family of kinases [Kannan et al., 2007
]. In addition to directly contributing to the catalytic efficacy of the enzyme, the C tail of some AGC kinases contains protein interaction motifs that recruit cellular regulatory factors. For example, the PXXP motif in PKCβII was shown to bind to two chaperone proteins (Hsp90 and cdc37) and contribute to protein stability [Gould et al., 2009
]. Moreover, a recent study identified a conserved FD(X)1–2
Y/D motif within the tail that is essential for docking of the activating kinase, PDK1 [Romano et al., 2009
]. Interestingly, in PKA, this motif encompasses Y330. In addition to modulating catatlytic activity itself, it is intriguing to speculate that phosphorylation of Y330 may also recruit the binding of a protein/s (e.g.
SH2 and/or PTB domain-containing proteins) to the C tail which would then add an entirely new element to the regulation of PKA activity. While additional experimentation will be required to determine the contributions of this phosphorylation to the spectrum of cellular events mediated by growth factors, the current results provide the mechanistic basis for crosstalk between growth factor RTKs and protein kinase A, two major signal transduction pathways.