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. 2014 Mar 4;53(8):1360-72.
doi: 10.1021/bi401329a. Epub 2014 Feb 19.

Structure and function of human DnaJ homologue subfamily a member 1 (DNAJA1) and its relationship to pancreatic cancer

Jaime L Stark  1 , Kamiya Mehla, Nina Chaika, Thomas B Acton, Rong Xiao, Pankaj K Singh, Gaetano T Montelione, Robert Powers
Affiliations

Structure and function of human DnaJ homologue subfamily a member 1 (DNAJA1) and its relationship to pancreatic cancer

Jaime L Stark et al. Biochemistry. .

Abstract

Pancreatic cancer has a dismal 5 year survival rate of 5.5% that has not been improved over the past 25 years despite an enormous amount of effort. Thus, there is an urgent need to identify truly novel yet druggable protein targets for drug discovery. The human protein DnaJ homologue subfamily A member 1 (DNAJA1) was previously shown to be downregulated 5-fold in pancreatic cancer cells and has been targeted as a biomarker for pancreatic cancer, but little is known about the specific biological function for DNAJA1 or the other members of the DnaJ family encoded in the human genome. Our results suggest the overexpression of DNAJA1 suppresses the stress response capabilities of the oncogenic transcription factor, c-Jun, and results in the diminution of cell survival. DNAJA1 likely activates a DnaK protein by forming a complex that suppresses the JNK pathway, the hyperphosphorylation of c-Jun, and the anti-apoptosis state found in pancreatic cancer cells. A high-quality nuclear magnetic resonance solution structure of the J-domain of DNAJA1 combined with a bioinformatics analysis and a ligand affinity screen identifies a potential DnaK binding site, which is also predicted to overlap with an inhibitory binding site, suggesting DNAJA1 activity is highly regulated.

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Figures

Figure 1
Figure 1
Protein sequence of DNAJA1 (UniProt entry P31689). The red residues are the 67 amino acids of the J-domain (DNAJA1-JD).
Figure 2
Figure 2
(A) Expression of full-length DNAJA1 (UniProt entry P31689) in MiaPaCa2 pancreatic cancer cells suppresses the activation of c-Jun in response to anisomycin (370 nM) or UV treatment (20 or 50 J/m2 doses). Twenty minutes after being treated, cells were subjected to lysis, and the levels of phospho-c-Jun (S63) and total c-Jun were evaluated by Western blotting. Expression levels of exogenously expressed His-tagged DNAJA1 were evaluated by immunoblotting with the anti-His antibody, while immunoblotting with the anti-tubulin antibody was performed as a loading control. (B) Expression of full-length DNAJA1 in MiaPaCa2 pancreatic cancer cells decreases the level of survival in response to anisomycin treatment-induced stress. The level of cell survival was measured by the MTT assay 24 h after treatment (**p < 0.01; ***p < 0.001).
Figure 3
Figure 3
(A) Overlay of the backbone trace of the 20 lowest-energy, water-refined structures. (B) Ribbon representation of the average structure generated from the average atomic coordinates of the 20 lowest-energy, water-refined structures, followed by water refinement of the average structure. Both structures are colored according to secondary structure: red for α-helix and white for loop.
Figure 4
Figure 4
Overlay of the ribbon structure for DNAJA1-JD (red) with (A) the E. coli DnaJ J-domain (PDB entry 1XBL), (B) the H. sapiens DnaJ homologue subfamily B member 1 J-domain (PDB entry 1HDJ), (C) H. sapiens DnaJ homologue subfamily B member 2 (PDB entry 2LGW), and (D) H. sapiens DnaJ homologue subfamily C member 12 (PDB entry 2CTQ). (E) ClustalW comparison of DNAJA1-JD (HR3099K) with PDB entries 1HDJ (blue), 1XBL (green), 2LGW (yellow), and 2CTQ (cyan). The highly conserved HPD sequence is outlined with a black box. The residues that make up helix α2 are outlined with a red box.
Figure 5
Figure 5
(A) Transparent surface and ribbon representation of DNAJA1-JD highlighting another proposed DnaK binding site based upon the bovine auxilin–bovine Hsp70 complex (PDB entry 2QWN) (colored red and green; conserved HPD motif). (B) Transparent surface and ribbon representation of DNAJA1-JD (rotated ∼90°) with the proposed DnaK binding site based upon NMR titration data (colored blue). (C) Transparent surface and ribbon of DNAJA1-JD (rotated ∼90°) with the proposed inhibition site based upon the TIM14–TIM16 complex (colored purple). (D) Sequence of DNAJA1-JD with the proposed interaction sites indicated: DnaK binding site from titrations (blue circle), DnaK inhibition site (purple triangles), DnaK binding site from the cross-linked auxilin–Hsp70 complex (red stars), and the highly conserved HPD motif (green box). The conserved HPD motif and helix α2, which is potentially an important component of the DnaJ–DnaK interaction site and the TIM16-like inhibitory binding site, are labeled.
Figure 6
Figure 6
(A) Overlay of 2D 1H–15N HSQC spectra of free DNAJA1-JD (black) and DNAJA1-JD with O-phospho-l-serine (red). (B) Transparent surface representation and ribbon diagram of DNAJA1-JD bound with O-phospho-l-serine, with the residues showing a chemical shift perturbation upon binding of O-phospho-l-serine colored blue and the one residue, Glu51, that shows the greatest chemical shift perturbation with every binding ligand colored red. (C) Expanded view of O-phospho-l-serine bound to a ribbon diagram of DNAJA1-JD, where residues with side chains directed toward the ligand are displayed.
Figure 7
Figure 7
Transparent surface representation and ribbon diagram of DNAJA1-JD bound with O-phospho-l-serine with (A) the proposed inhibition site based on the TIM14–TIM16 interaction (purple), (B) the highly conserved (magenta) and poorly conserved (cyan) residues from Consurf, and (C) the positively charged surface (blue) and negatively charged surface (red) from Delphi. Helix α2, which is potentially an important component of the DnaJ–DnaK interaction site and the TIM16-like inhibitory binding site, is labeled.
Figure 8
Figure 8
Illustration of the proposed role of DNAJA1 and DnaK on the JNK pathway and c-Jun phosphorylation. The activation of DNAJA1 through the interaction with DnaK appears to suppress the JNK pathway, thus keeping c-Jun in the inactive state. However, inhibiting binding of DNAJA1 to DnaK, as TIM16 does, would allow for the hyperphosphorylation of c-Jun, which has an anti-apoptotic effect.
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