Interleukin-3 (IL-3) is a multipotential hematopoietic growth factor (HGF) that mediates growth and differentiation of target cells by triggering post- receptor signaling resulting in the activation of key intermediate protein kinases (Fig. 1). Implicit in growth factor action, however, is the requirement for the synthesis of structural and regulatory proteins necessary for growth. Thus, efficient growth results from the coordinated stimulation of proliferation and protein synthesis as well as the inhibition of apoptosis. Little is known about how IL-3 might couple to and activate protein synthesis. Preliminary data indicate that IL-3 reversibly affects protein synthesis in a novel signaling pathway featuring the rapid regulation of the double stranded RNA activatable protein kinase, PKR. PKR is an INF-inducible gene well known to play a role in viral-induced inhibition of protein synthesis as a part of the host-cell antiviral defense mechanism. PKR is ubiquitously expressed and has recently been found to have potential tumor-suppressor properties. Furthermore, activated Ha-ras transformed 3T3 cells contain a specific inhibitor of PKR. These findings suggest a necessary role for PKR in cell growth. The applicants find that IL-3 withdrawal mediates phosphorylation and enzymatic activation of PKR with phosphorylation of its physiologic substrate, eIF2a. When phosphorylated, eIF2a is known to inhibit initiation of protein synthesis (Fig 2). Alternatively, IL-3 addition to factor-deprived cells mediates dephosphorylation, and inactivation of PKR is closely associated with the rapid interaction of PKR and a 97 kDa tyrosine and serine-containing phosphoprotein, pp97. IL-3 can regulate PKR during normal growth in addition to its well recognized role in antiviral host defense. In order to elucidate the molecular features of this novel IL-3 pathway, the investigator will focus on how IL-3 reversibly regulates PKR and protein synthesis.
The specific aims are to determine: (1) the mechanism by which IL-3 withdrawal activates PKR and inhibits protein synthesis; (2) the mechanism by which IL-3 addition to deprived cells can inactivate PKR and derepress protein synthesis; and (3) to purify, characterize, molecularly clone and express pp97. State of the art molecular and biochemical methodology will be employed, including construction and enforced expression of catalytically inactive PKR, antisense technology, and use of the yeast two-hybrid system for interactive cloning of pp97. It is expected that PKR may represent a unique therapeutic target for novel anti-neoplastic strategies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL054083-03
Application #
2685442
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1996-04-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
1999-03-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Cheng, Xiaodong; Byrne, Michael; Brown, Kevin D et al. (2015) PKR inhibits the DNA damage response, and is associated with poor survival in AML and accelerated leukemia in NHD13 mice. Blood 126:1585-94
Byrne, Michael; Bennett, Richard L; Cheng, Xiaodong et al. (2014) Progressive genomic instability in the Nup98-HoxD13 model of MDS correlates with loss of the PIG-A gene product. Neoplasia 16:627-33
Cheng, X; Bennett, R L; Liu, X et al. (2013) PKR negatively regulates leukemia progression in association with PP2A activation, Bcl-2 inhibition and increased apoptosis. Blood Cancer J 3:e144
Liu, Xiangfei; Bennett, Richard L; Cheng, Xiaodong et al. (2013) PKR regulates proliferation, differentiation, and survival of murine hematopoietic stem/progenitor cells. Blood 121:3364-74
Bennett, Richard L; Pan, Yu; Christian, Jaime et al. (2012) The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G? arrest. Cell Cycle 11:407-17
Bennett, Richard L; Carruthers, Aubrey L; Hui, Teng et al. (2012) Increased expression of the dsRNA-activated protein kinase PKR in breast cancer promotes sensitivity to doxorubicin. PLoS One 7:e46040
Bennett, Richard L; Blalock, William L; Choi, Eun-Jung et al. (2008) RAX is required for fly neuronal development and mouse embryogenesis. Mech Dev 125:777-85
Bennett, Richard L; Blalock, William L; Abtahi, Dean M et al. (2006) RAX, the PKR activator, sensitizes cells to inflammatory cytokines, serum withdrawal, chemotherapy, and viral infection. Blood 108:821-9
Bennett, Richard L; Blalock, William L; May, W Stratford (2004) Serine 18 phosphorylation of RAX, the PKR activator, is required for PKR activation and consequent translation inhibition. J Biol Chem 279:42687-93
Varma, Tushar; Liu, Si Qi; West, Matthew et al. (2003) Protein kinase C-dependent phosphorylation and mitochondrial translocation of aldose reductase. FEBS Lett 534:175-9

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