A higher order of regulation can be achieved by compartmentalization and functional specialization of the endocytic pathway during signal transduction in response to growth factors (GFs). In contrast, impairment of intracellular (IC) trafficking of GF signaling complexes can result in mis-localization or accumulation of signaling molecules in the wrong compartment, or at the wrong time, giving rise to various disease states, including cancer. In addition, the specificity of motor protein light chains (LCs) in recruiting their membranous cargo for IC transport during regulation of GF signaling is becoming increasingly clear. Additional investigation of the regulation, key components, timing, and functional outcome of signaling from endosomal subcompartments is required to enable oncology drug development, based upon these newer signaling regimes. Our data from the last project period indicated that km23 dynein LCs are activated by TGF? receptors (T?Rs) and are required for Smad trafficking, prior to nuclear translocation. Thus, km23 appears to function as a """"""""motor receptor"""""""" to recruit TGF? signaling complexes to the dynein motor for IC transport of cargo (ie, T?Rs, Smads) along microtubules (MTs) toward the nucleus. In addition, we have identified another related member of the km23/LC7/robl/DYNLRB family, termed km23-2. While this isoform appears to have some characteristics similar to km23-1, km23-2 appears to more specifically regulate Smad3, in contrast to Smad2 regulation by km23-1. The mechanisms underlying the differential regulation and trafficking of Smads2/3 in relation to the km23-1/2 motor LCs will be explored in the new application, including how km23 impacts downstream TGF? responses. During the last project period, we have also shown that protein kinase A (PKA) directly phosphorylates km23 and is required for km23 binding to the dynein intermediate chain (DIC). The mechanisms underlying this PKA phosphorylation of km23 in TGF? signaling and Smad2/3 trafficking will also be examined. Additional studies relate to the investigation of how these trafficking events are altered in human ovarian cancer cells (HOCCs) known to express altered forms of km23-1. As part of the last project period, we identified km23-1 alterations in 42% of ovarian cancer patient tissues, with no km23 alterations detectable in normal tissues. Such a high alteration rate in ovarian cancer suggests that km23 may play an important role in either TGF2 resistance or tumor progression of this disease. In the new application, we will examine the effects of knocking down km23, or mutating it at key phosphorylation sites, on Smad trafficking, Smad-specific TGF? responses, and the malignant phenotype of the human cancer cells in vitro, as well as on tumor progression in vivo.
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