The Fanconi Anemia Proteins Functionally Interact with the Protein Kinase Regulated by RNA (PKR)

Xiaoling Zhang,June Li,Daniel P Sejas,Keaney R Rathbun,Grover C Bagby,Qishen Pang

doi:10.1074/jbc.m403884200

Abstract

Protein kinase regulated by RNA (PKR) plays critical roles in cell growth and apoptosis and is implicated as a potential pathogenic factor of Alzheimer's, Parkinson's, and Huntington's diseases. Here we report that this proapoptotic kinase is also involved in Fanconi anemia (FA), a disease characterized by bone marrow (BM) failure and leukemia. We have used a BM extract to show that three FA proteins, FANCA, FANCC, and FANCG, functionally interact with the PKR kinase, which in turn regulates translational control. By using a combined immunoprecipitation and reconstituted kinase assay, in which an active PKR kinase complex was captured from a normal cell extract, we demonstrated functional interactions between the FA proteins and the PKR kinase. In primary human BM cells, mutations in the FANCA, FANCC, and FANCG genes markedly increase the amount of PKR bound to FANCC, and this PKR accumulation is correlated with elevated PKR activation and hypersensitivity of BM progenitor cells to growth repression mediated by the inhibitory cytokines interferon-gamma and tumor necrosis factor-alpha. Specific inhibition of PKR by 2-aminopurine in these FA BM cells attenuates PKR activation and apoptosis induction. In lymphoblasts derived from an FA-C patient, overexpression of a dominant negative mutant PKR (PKRK296R) suppressed PKR activation and apoptosis induced by interferon-gamma and tumor necrosis factor-alpha. Furthermore, by using genetically matched wild-type and PKR-null cells, we demonstrated that forced expression of a patient-derived FA-C mutant (FANCCL554P) augmented double-stranded RNA-induced PKR activation and cell death. Thus, inappropriate activation of PKR as a consequence of certain FA mutations might play a role in bone marrow failure that frequently occurred in FA.

Highlights

The Protein kinase regulated by RNA (PKR)1 plays a critical role in translational control [1]
Mutations in these Fanconi anemia (FA) genes cause abnormal accumulation of PKR kinase bound to the FANCC protein, which is correlated with elevated PKR activation and hypersensitivity of bone marrow (BM) progenitor cells to growth repression mediated by inhibitory cytokines IFN-␥ and TNF-␣
The increased association of the PKR kinase with the FANCC protein in lymphoblasts derived from an FA patient carrying an FANCC mutation [23] prompted us to examine the association of FANCC, PKR, and Hsp70 in cells from FA patients bearing mutations in other FA genes

Summary

EXPERIMENTAL PROCEDURES

In Vivo 32P and 35S Labeling of Proteins—Cells were starved for 60 min in phosphate-free medium containing 10% dialyzed fetal bovine serum and were treated with a combination of IFN-␥ and TNF-␣ (10 ng/ml each, R & D Systems) for 2 h. Cells were rinsed with methionine-cysteine-free medium, and labeling was again performed in the same medium containing 50 ␮Ci/ml [35S]methioninecysteine labeling mix (PerkinElmer Life Sciences) and IFN-␥ and TNF-␣ (10 ng/ml each, R & D Systems) and incubated at 37 °C for an additional hour. Cells were harvested, washed with phosphate-buffered saline, and resuspended at a concentration of 1 ϫ 106/ml in staining buffer containing phosphate-buffered saline, 2% fetal calf serum, 0.1% sodium azide This cell suspension (400 ␮l) was added to 400 ␮l of Cytofix/Cytoperm (Pharmingen) and incubated for 20 min on ice to fix and permeabilize the cells.

FA Proteins Interact with PKR Kinase

To confirm the contribution of PKR activation to the FA

DISCUSSION