Regulation of human protein kinases and phosphatases by 14-3-3
The first part of my thesis shows that a previously-uncharacterised protein FAM122A and its paralogue FAM122C form stable stoichiometric complexes with the protein phosphatase 2A (PP2A) heterotrimer (AαB55αC). Moreover, the FAM122A–PP2A complex is active and contains methylated catalytic C subunit. In contrast, a complex comprising the protein phosphatase methylesterase-1 (PME-1) and the C-subunit of PP2A does not interact with FAM122A. I also identified that FAM122A and its paralogues FAM122B and FAM122C are 14-3-3-binding phosphoproteins. The binding of 14-3-3 to FAM122A is mediated by phosphorylation of Ser37 and Ser62; the latter being a site that is phosphorylated in response to cAMP-elevating agents. Binding of 14-3-3 does not affect the interaction of FAM122A with PP2A. Rather, binding of 14-3-3 promotes localisation of FAM122A to the nucleus, perhaps by masking the nuclear export signal (NES) that I identified. Another possibility is that 14-3-3 binding exposes a nuclear localisation signal (NLS) that was identified on FAM122A, and whose mutation promotes cytoplasmic localisation of the protein. The second part of my thesis stems from my confirmation of bioinformatic predictions that several protein kinases, including MINK1, MAST4, TAOK1, CLK1 and the group II PAK kinases PAK4, PAK6 and PAK7 are novel 14-3-3 binding phosphoproteins. The binding of 14-3-3 to group II PAK kinases is differentially regulated by various signal transduction pathways. The phosphorylation of Ser99 and Ser181 in response to phorbol ester mediates 14-3-3 binding to PAK4. 14-3-3 binding localises PAK4 to the leading edge of the cell. Moreover, Ser/Thr99 is a ‘lynchpin’ site that is conserved across the group II PAK family. In contrast to PAK4, cAMP elevating agents and insulin-like growth factor 1 (IGF1) stimulate phosphorylation of Ser113 that mediates 14-3-3 binding to PAK6. The binding to 14-3-3s retains PAK6 in the cytoplasm, while upon mutation of 14-3-3 binding sites PAK6 is localised to cell-cell junctions. Epidermal growth factor (EGF) and cAMP elevating agents stimulate 14-3-3 binding to PAK7. Further results suggest that PAK4, and not PAK6, has a predominant role in regulating cell proliferation, adhesion and migration of melanoma cells carrying B-RAFV600E and N-RASQ61K mutations possibly by phosphorylating substrates such as LIMK1 and GEF-H1. PAK7 shares common substrates with PAK4 and probably has a redundant role in regulating migration of melanoma cells. Overall, the work in this thesis gives a broader view on integration of the complex networks of signaling pathways. My study also serves as an example of how 14-3-3 binding regulates the function of protein kinases and phosphatases in cells.
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