In addition to the kinase required to phosphorylate the binding motif, the ability of 14-3-3 proteins to interact with a target can be regulated by other inhibitory kinases. In chapter 3, dematin was identified as a novel substrate of the p38 MAPK, and we therefore aimed to investigate whether p38 plays a regulatory role in the interaction between dematin and 14-3-3β. This was of particular interest as the 14- 3-3 binding motifs at Ser22, Ser85, and Ser269 that were identified in the mass spectrometry analysis all contain closely neighbouring or overlapping potential p38 phosphorylation motifs.
4.5.1 The dematinC1/ 14-3-3β interaction is not affected by p38 phosphorylation The effect of p38 phosphorylation was first tested on the known 14-3-3 binding sites at Ser269 and Ser333, with single serine-alanine mutants used to look at the regulation of each site individually. Thr274 is a potential p38 phosphorylation motif that neighbours the Ser269 14-3-3 binding motif (Figure 4.10A), and this was identified as being phosphorylated in our mass spectrometry (Chapter 3, Figure 3.5). The GST pulldown experiment between pDEST27FLAG-dematinC1_S269A or pDEST27FLAG-dematinC1_S333A and pcDNA3.1MYC-14-3-3β were repeated under
142 Figure 4.10: Alterations in p38 phosphorylation state do not affect the interaction between 14-3-3β and the C-terminal of dematin
HEK293T cells were co-transfected with pDEST27FLAG-dematin_S269A or pDEST27FLAG-dematin_S333A, and pcDNA3.1MYC-14-3-3β. For p38 activation cells were co-transfected with pcDNA3FLAG-MKK6. After 48 hours, p38 inhibited cells were treated with the specific inhibitor SB203580 for 4 hours. Cells lysates were then purified on GST-pulldown columns, separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were immunoblotted with anti-FLAG and anti-MYC antibodies. (GST; glutathione S-transferase, wcl; whole cell lysate).
untreated p38 activated p38 inhibited
wcl GST wcl GST wcl GST
C1-S333A 14-3-3β
untreated p38 activated p38 inhibited
wcl GST wcl GST wcl GST C1-269A 14-3-3β
Q R G R M D R G N S L P C V L E Q K
K S L P I R R K T R S L P D R T P F H
269 333A
B
C
143 conditions of varying p38 phosphorylation status. p38 was hyper-activated through co-transfected with pcDNAFLAG-MKK6DD, a specific upstream activator, or inhibited with a 4 hour incubation with the specific inhibitor SB203580 immediately prior to cell lysis. Alterations in p38 phosphorylation state were insufficient to prevent an interaction between dematinC1 and 14-3-3β at the RKTRS269LP (Figure 4.10B) motif or at the RGNS333LP motif (Figure 4.10C). This suggests either that these sites are not being phosphorylated by p38, or that p38 phosphorylation of these neighbouring sites does not inhibit dematin / 14-3-3 association.
4.5.2 p38 phosphorylation does not regulate an N-terminal / 14-3-3β interaction The N-terminal 14-3-3 binding motifs at Ser22 and Ser85 were unable to mediate an interaction between dematin and 14-3-3. Here we aimed to investigate whether p38 phosphorylation of dematin was inhibiting this interaction by preventing the association of 14-3-3β. Residues Ser16, Ser26, Ser87 and Ser92 were all phosphorylated in our mass spectrometry analysis of dematin (Chapter 3, Figure 3.5). As can be seen in Figure 4.11A, these phosphorylation sites either closely neighbour or overlap the 14-3-3 binding motif. The co-immunoprecipitation experiments between pDEST27FLAG-dematinN1 or dematinN3 and pcDNA3.1MYC- 14-3-3β were repeated with co-transfection of pcDNAFLAG-MKK6DD or incubation with SB203580 inhibitor. No co-immunoprecipitation of 14-3-3β was seen with either dematinN1 (Figure 4.11B) or dematinN3 (Figure 4.11C) under p38 hyper- activated or inhibition conditions. This suggests that the absence of a detectable interaction between 14-3-3β and dematin at the RDSS22VP or RERS85LSP motifs is not due to p38 phosphorylating neighbouring sites and hindering 14-3-3 binding. GST-pulldown experiments were conducted using HEK293T cells co-transfected with pDEST27FLAG-dematinN1_S16/26A or pDEST27FLAG-dematinN3_S87/92A and pcDNA3.1MYC-14-3-3β. Co-purification of 14-3-3β was not detected with either the dematinN1_S16/26A (Figure 4.12B), nor dematinN3_S87/92A (Figure 4.12C), constructs. This indicates that the absence of a detectable interaction between dematin and 14-3-3β at the RDSS22VP and RERS85LSP motifs is not due to the inhibitory phosphorylation of the neighbouring Ser16/ Ser26 and Ser87 / Ser92 residues.
144 Figure 4.11: Alterations in p38 phosphorylation state are not sufficient to induce and interaction between 14-3-3β and the N-terminal of dematin
HEK293T cells were co-transfected with pDEST27FLAG-dematinN1 or dematinN3, and pcDNA3.1MYC-14-3-3β. For p38 hyper-activation, cells were co-transfected with pcDNA3FLAG-MKK6. After 48 hours, p38 inhibited cells were treated with the specific inhibitor SB203580 for 4 hours. Cells lysates were then purified on GST- pulldown columns, separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were immunoblotted with anti-FLAG and anti-MYC antibodies. (GST; glutathione S-transferase, wcl; whole cell lysate).
untreated p38 activated p38 inhibited
wcl GST wcl GST wcl GST
N1 14-3-3β
untreated p38 activated p38 inhibited
wcl GST wcl GST wcl GST N3 14-3-3β
22
G S V S P S R D S S V P G S P S S I
85
V E L P R S R E R S L S P K S T S P
A
B
C
145 Figure 4.12: Phosphorylation of neighbouring residues does not prevent and N- terminal dematin / 14-3-3β interaction
(A) Site-directed mutagenesis was used to change serine residue neighbouring 14-3- 3 motifs to alanine residues. HEK293T cells were co-transfected with (B) pDEST27FLAG-dematinN1 / pDEST27FLAG-dematinN1_S11/16A or (C) pDEST27FLAG-dematinN3 / pDEST27FLAG-dematinN3_S87/92A, and pcDNA3.1MYC-14-3-3β. Cells lysates were purified on GST-pulldown columns, separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were immunoblotted with anti-FLAG and anti-MYC antibodies. (GST; glutathione S- transferase, wcl; whole cell lysate).
16 22 G S V A P S R D S S V P G A P S S I 85 87 V E L P R S R E R S L A P K S T A P 26 92
N1
N1_S11/16A
wcl GST wcl GST N1 14-3-3βN3
N3_S87/92A
wcl GST wcl GST N3 14-3-3βB
C
A
146