The phosphorylated, activated cytoplasmic domains of the transforming growth factor-␤ (TGF␤) receptors were used as probes to screen an expression library that was prepared from a highly TGF␤-responsive intestinal epithelial cell line. One of the TGF␤ receptor-interacting proteins isolated was identified to be the mammalian homologue of the LC7 family (mLC7) of dynein light chains (DLCs). This 11-kDa cytoplasmic protein interacts with the TGF␤ receptor complex intracellularly and is phosphorylated on serine residues after ligand-receptor engagement. Forced expression of mLC7-1 induces specific TGF␤ responses, including an activation of Jun N-terminal kinase (JNK), a phosphorylation of c-Jun, and an inhibition of cell growth. Furthermore, TGF␤ induces the recruitment of mLC7-1 to the intermediate chain of dynein. A kinase-deficient form of TGF␤ RII prevents both mLC7-1 phosphorylation and interaction with the dynein intermediate chain (DIC). This is the first demonstration of a link between cytoplasmic dynein and a natural growth inhibitory cytokine. Furthermore, our results suggest that TGF␤ pathway components may use a motor protein light chain as a receptor for the recruitment and transport of specific cargo along microtublules. INTRODUCTIONTransforming growth factor-␤ (TGF␤) is the prototype for the TGF␤ superfamily of highly conserved growth regulatory polypeptides that also includes the activins, inhibins, bone morphogenetic proteins, decapentaplegic (Dpp), nodal, Lefty, and others (Roberts, 1998;Sporn and Vilcek, 2000;Yue and Mulder, 2001). Alterations in the TGF␤ signaling components and pathways have been implicated in a vast array of human pathologies, including cancer (Massague et al., 2000;Sporn and Vilcek, 2000;Derynck et al., 2001).TGF␤ binds to two types of transmembrane serine/threonine kinase receptors (RI and RII) in a heterotetrameric complex, to activate downstream components (Roberts, 1998; Massague et al., 2000;Sporn and Vilcek, 2000;Yue and Mulder, 2001). The Smad family of signaling intermediates plays an important role in mediating TGF␤ responses (Attisano and Wrana, 2000;ten Dijke et al., 2000;Yue and Mulder, 2001). Moreover, TGF␤ has been shown to regulate Ras (Mulder and Morris, 1992;Hartsough et al., 1996;Yue et al., 1998) and several components of the mitogen-activated protein kinase (Mapk) pathways (Hartsough and Mulder, 1995;Frey and Mulder, 1997;Mulder, 2000;Sporn and Vilcek, 2000;Yue and Mulder, 2001). In addition to the Ras/Mapk and Smad pathways, several proteins have been identified based upon their interaction with the TGF␤ receptors (Yue and Mulder, 2001). Furthermore, various Smad-interacting proteins have also been identified, including SARA and Dab2, which interact with both Smads and the TGF␤ receptors (Tsukazaki et al., 1998;Hocevar et al., 2001;Yue and Mulder, 2001).Despite advances in our understanding of the mechanisms by which the Smad and Ras/Mapk cascades mediate some TGF␤ effects, these pathways seem to regulate primarily transcriptional events (Hocevar et al., 1...

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm.

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km23-1 was originally identified as a TGFβ receptor-interacting protein that plays an important role in TGFβ signaling. Moreover, km23-1 is actually part of an ancient superfamily of NTPase-regulatory proteins, widely represented in archaea and bacteria. To further elucidate the function of km23-1, we identified novel protein interacting partners for km23-1 by using tandem affinity purification (TAP) and tandem mass spectrometry (MS). Here we show that km23-1 interacted with a class of proteins involved in actin-based cell motility and modulation of the actin cytoskeleton. We further showed that km23-1 modulates the formation of a highly organized stress fiber network. More significantly, we demonstrated that knockdown (KD) of km23-1 decreased RhoA activation in Mv1Lu epithelial cells. Finally, our results demonstrated for the first time that depletion of km23-1 inhibited cell migration of human colon carcinoma cells (HCCCs) in wound-healing assays. Overall, our findings demonstrate that km23-1 regulates RhoA and motility-associated actin modulating proteins, suggesting that km23-1 may represent a novel target for anti-metastatic therapy.

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