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.
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