Purification of protein phosphatases and 14-3-3-binding proteins and their regulation by the cAMP–PKA pathway

Abstract

Reversible protein phosphorylation is a highly conserved mechanism that regulates the majority of proteins in a eukaryotic cell. Protein phosphorylation has been implicated in various cellular processes including glycogen metabolism, cell cycle control, RNA splicing, DNA synthesis, muscle contraction and mitosis. Protein kinases and phosphatases regulate the phosphorylation status of proteins in response to intra and extracellular stimuli such as hormones and growth factors.

In chapter 3, I explore the regulation of protein phosphatases when the cAMP–PKA signalling pathway is activated and study the isoform specificity of PP1. Most protein phosphatases of the PPP family do not exist as free catalytic subunits: instead, they interact with regulatory subunits, which specify, target and/or inhibit their activities. Protein phosphatases of the PPP family such as PP1 and PP2, act specifically on serine/threonine phosphorylations; our understanding of phosphatase regulation stems from the regulation of members of the PPP family upon activation of the cAMP signalling pathway through protein kinase A. We wanted to find out whether there were more proteins that would regulate phosphatase activity when the cAMP–PKA pathway is activated and so started by doing a microcystin-affinity purification. Microcystin is a potent inhibitor of most PPP phosphatases and has been adapted to purify protein phosphatases from cellular extracts. I used the cAMP agonist forskolin to trigger activation of the cAMP–PKA pathway and used the H-89 inhibitor to block PKA activity. Although several protein phosphatases were identified along with their regulatory subunits, I could not find any striking enrichment of proteins upon activation or inhibition of the cAMP–PKA signalling pathway.

I also purified protein phosphatase 1 complexes from a cell line stably expressing GFP-TAP- PP1β with the aim of identifying PP1-regulatory subunits which would indicate potential PKA-mediated regulatory events. Again, cells were stimulated with forskolin and H-89 to monitor changes in the cAMP–PKA signalling pathway. Many known PP1 interacting proteins were identified including MYPT1, NIPP1, Inhibitor-2 and Staufen. Interestingly, three glycogen-related proteins, namely R6, glycogenin and glycogen synthase, were found to be highly enriched upon forskolin treatment which included prior inhibition of the pathway with H-89. R6 is a glycogentargeting subunit of PP1 while glycogenin acts as the primer during glycogen synthesis; these glycogen moieties are then elongated by glycogen synthase. We think the enrichment of glycogen synthase and glycogenin from PP1 immunoprecipitates treated with H-89 and forskolin could be linked to increased amounts of the R6-glycogen targeting subunit under the same conditions. Further experiments are required to check the correlation between R6 and the enzymes of glycogen synthesis.

PP1 isoforms alpha, beta and gamma are 97% identical in their catalytic residues, raising an important question of how these isoforms exert specificity in their substrates. Isoform specificity has been implicated in a number of regulatory processes including the selective nuclear targeting of the PP1γ subunit by Repo-Man and the preferential association of MYPT1 with PP1β to control muscle contraction. With such a high degree of similarity among PP1 isoforms, I wanted to evaluate whether the three isoforms varied much in their isoform specificity or whether they could be redundant in their substrates. For this, I stably expressed the three isoforms with an N-terminal GFPTAP tag and analysed the immunoprecipitated protein complexes for each isoform. We found that there was no significant pattern of enrichment for any protein that could be used to specify isoform preference. Nonetheless, we did find higher amounts of MYPT1 with the beta isoform compared to alpha and gamma, in agreement with the reports of preferential PP1 beta binding to the ankyrin repeats of MYPT1. Overall though, these studies suggest that isoform specificity of the PP1 catalytic subunits may not be clear cut; in fact, most studies reporting PP1 isoform selectivity use bacterially-expressed PP1 proteins which have been shown to exhibit different properties to the native enzymes.

Protein phosphorylation creates binding sites for proteins such as 14-3-3s, which have been shown to dock onto dually-phosphorylated serine/threonine residues on target proteins. In chapter 4, I looked at the regulation of 14-3-3-binding proteins when the cAMP–PKA signalling pathway was activated. Again, I used forskolin as a cAMP agonist and H-89 as the non-specific PKA inhibitor to monitor changes in proteins involved in 14-3-3-binding in this pathway. 14-3-3s are involved in regulating various cellular proteins involved in insulin signalling, transcription, translation, glycogen metabolism, autophagy, vesicle and protein trafficking among others. Previous experiments in our laboratory explored the binding of 14-3-3s to its targets upon stimulation with hormones and growth factors such as insulin, IGF-1 and EGF, to monitor how changes in these pathways altered proteins involved in survival and growth promoting pathways. Understanding how and when 14-3-3s impact on these signalling targets enables us to gain regulatory insights into many areas of 14-3-3 biology. Since many proteins had already been observed to bind 14-3-3s upon forskolin stimulation, I performed a large-scale 14-3-3 affinity pull-down to explore proteins in the cAMP–PKA proliferation-promoting pathway. Interestingly, many forskolin/H-89-regulated proteins were identified and I went on to validate a few of these candidate proteins as 14-3-3-binding targets, including the DENND4 family, EML3 and Atg9. The DENND4 proteins are guanine nucleotide exchange factors for Rab proteins, while Atg9 is a component of the autophagosome. Therefore, my findings indicate novel roles for 14-3-3s in regulation of intracellular membrane dynamics. My studies indicate that there are still many targets of 14-3-3s to be characterised and I hope that the data presented in my thesis will be extended in future and contribute towards our understanding of protein phosphorylation and14-3-3 biology.

Date of Award	2012
Original language	English
Sponsors	The James Hutton Institute & Medical Research Council
Supervisor	Carol MacKintosh (Supervisor)