Qunyan Jin

Requirement for the Dynein Light Chain km23-1 in a Smad2-dependent Transforming Growth Factor-β Signaling Pathway

We have identified km23-1 as a novel transforming growth factor-β (TGFβ) receptor (TβR)-interacting protein that is also a light chain of the motor protein dynein (dynein light chain). Herein, we demonstrate by sucrose gradient analyses that, in the presence of TGFβ but not in the absence, km23-1 was present in early endosomes with the TβRs. Further, confocal microscopy studies indicate that endogenous km23-1 was co-localized with endogenous Smad2 at early times after TGFβ treatment, prior to Smad2 translocation to the nucleus. In addition, immunoprecipitation/blot analyses showed that TGFβ regulated the interaction between endogenous km23-1 and endogenous Smad2 in vivo. Blockade of km23-1 using a small interfering RNA approach resulted in a reduction in both total intracellular Smad2 levels and in nuclear levels of phosphorylated Smad2 after TGFβ treatment. This decrease was reversed by lactacystin, a specific inhibitor of the 26 S proteasome, suggesting that knockdown of km23-1 causes proteasomal degradation of phosphorylated (i.e. activated) Smad2. Blockade of km23-1 also resulted in a reduction in TGFβ/Smad2-dependent ARE-Lux transcriptional activity, which was rescued by a km23-1 small interfering RNA-resistant construct. In contrast, a reduction in TGFβ/Smad3-dependent SBE2-Luc transcriptional activity did not occur under similar conditions. Furthermore, overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocked TGFβ-stimulated nuclear translocation of Smad2. Collectively, our findings indicate for the first time that a dynein light chain is required for a Smad2-dependent TGFβ signaling pathway.

Role of km23-1 in RhoA/actin-based cell migration

km23-1 was originally identified as a TGFß receptor-interacting protein that plays an important role in TGFß 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.

The TGFβ receptor-interacting protein km23-1/DYNLRB1 plays an adaptor role in TGFβ1 autoinduction via its association with Ras

Background: The TGFβ receptor-interacting protein km23-1 plays an important role in TGFβ signal transduction in TGFβ-sensitive epithelial cells. Results: The role of km23-1 in TGFβ activation of Ras/JNK/ERK, as well as TGFβ1 autoinduction, was determined. Conclusion: km23-1 is required for TGFβ1 autoinduction through a Smad2-independent Ras/ERK/JNK pathway. Significance: km23-1 functions as a critical adaptor coupling TGFβ receptors to Ras activation after TGFβ treatment. We have previously elucidated the signaling events that are required for TGFβ1 autoinduction (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765–30773). Further, we have reported that the TGFβ receptor (TβR)-interacting protein km23-1 plays an important role in TGFβ signal transduction (Jin, Q., Ding, W., and Mulder, K. M. (2007) J. Biol. Chem. 282, 19122–19132). Here we examined the role of km23-1 in TGFβ1 autoinduction in TGFβ-sensitive epithelial cells. siRNA blockade of km23-1 reduced TGFβ1 mRNA expression, as well as DNA binding and transcriptional activation of the relevant activator protein-1 site in the human TGFβ1 promoter. Further, knockdown of km23-1 inhibited TGFβ-mediated activation of ERK and JNK, phosphorylation of c-Jun, and transactivation of the c-Jun promoter. Sucrose gradient analyses indicate that km23-1 was present in lipid rafts together with Ras and TβRII after TGFβ treatment. Immunoprecipitation/blot analyses revealed the formation of a TGFβ-inducible complex between Ras and km23-1 in vivo within minutes of TGFβ addition. Moreover, we demonstrate for the first time that km23-1 is required for Ras activation by TGFβ. Our results indicate that km23-1 is required for TGFβ1 autoinduction through Smad2-independent Ras/ERK/JNK pathways. More importantly, our findings demonstrate that km23-1 functions as a critical adaptor coupling TβR activation to activation of Ras effector pathways downstream.

Requirement of a dynein light chain in transforming growth factor β signaling in zebrafish ovarian follicle cells.

We have previously reported that the dynein light chains km23-1 and km23-2 are required for TGFβ signaling in mammalian cells. Here we describe another member of the km23/DYNLRB/LC7/robl family of dynein light chains in zebrafish, termed zkm23, which is also involved in TGFβ signaling. zkm23 was rapidly phosphorylated after TGFβ stimulation. TGFβ RII kinase activity was absolutely required for zkm23 phosphorylation, whereas a constitutively active TGFβ RI did not induce phosphorylation. Further, TGFβ stimulated a rapid recruitment of the zkm23 dynein light chain to the dynein intermediate chain of the dynein complex, and the TGFβ RII kinase was required for this interaction. Finally, blockade of zkm23 using morpholino oligos resulted in an inhibition of TGFβ-mediated transcriptional responses. Thus, our results demonstrate for the first time that the dynein light chain zkm23 is required for TGFβ signaling in cultured zebrafish ovarian follicle cells.

Overexpression of the dynein light chain km23-1 in human ovarian carcinoma cells inhibits tumor formation in vivo and causes mitotic delay at prometaphase/metaphase

km23‐1 is a dynein light chain that was identified as a TGFβ receptor‐interacting protein. To investigate whether km23‐1 controls human ovarian carcinoma cell (HOCC) growth, we established a tet‐off inducible expression system in SKOV‐3 cells in which the expression of km23‐1 is induced upon doxycycline removal. We found that forced expression of km23‐1 inhibited both anchorage‐dependent and anchorage‐independent growth of SKOV‐3 cells. More importantly, induction of km23‐1 expression substantially reduced the tumorigenicity of SKOV‐3 cells in a xenograft model in vivo. Fluorescence‐activated cell sorting analysis of SKOV‐3 and IGROV‐1 HOCCs demonstrated that the cells were accumulating at G2/M. Phospho‐MEK, phospho‐ERK and cyclin B1 were elevated, as was the mitotic index, suggesting that km23‐1 suppresses HOCCs growth by inducing a mitotic delay. Immunofluorescence analyses demonstrated that the cells were accumulating at prometaphase/metaphase with increases in multipolar and multinucleated cells. Further, although the mitotic spindle assembly checkpoint protein BubR1 was present at the prometaphase kinetochore in Dox+/− cells, it was inappropriately retained at the metaphase kinetochore in Dox− cells. Thus, the mechanism by which high levels of km23‐1 suppress ovarian carcinoma growth in vitro and inhibit ovary tumor formation in vivo appears to involve a BubR1‐related mitotic delay.

A dynein motor attachment complex regulates TGFß/Smad3 signaling.

Our previous results have demonstrated that km23-2 has functions in TGFß signaling that are distinct from those for km23-1. In the current report, we demonstrate that blockade of km23-2 decreased TGFß activation of the human Smad7 promoter Smad7-Luc, an endogenous Smad3-target promoter. Luminescence-based mammalian interaction mapping (LUMIER) analyses showed that TGFß stimulated the interaction of km23-2 preferentially with Smad3, relative to that with Smad2. Size exclusion chromatography experiments revealed that km23-2 and Smad3 were recruited into the same complex after TGFß treatment. Moreover, in the presence of TGFß, but not in the absence, km23-2 was present in early endosomes with the TGFß receptors (TßRs) and Smad3. Collectively, our data indicate that km23-2 is a critical signaling intermediate in a Smad3-dependent TGFß signaling pathway. We also provide evidence of the novel finding that TGFß stimulates the rapid recruitment of the km23-2 dimer to the dynein intermediate chain (DIC) of the dynein complex, whereas a kinase-deficient form of TßRII prevented this interaction. Finally, we demonstrate for the first time that TGFß stimulated not only assembly of the dynein motor attachment complex, but also triggered the tethering of the km23-2-Smad3 cargo to the other dynein components. Thus, our data demonstrate a novel function for km23-2 as a motor receptor to recruit Smad3 to the dynein complex for intracellular transport, thereby mediating Smad3-dependent TGFß signaling.

Requirement for protein kinase A in the phosphorylation of the TGFβ receptor-interacting protein km23-1 as a component of TGFβ downstream effects

km23-1 was previously identified as a TGFβ-receptor interacting protein that was phosphorylated on serines after TGFβ stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFβ/protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites. In vitro kinase assays further demonstrated that PKA directly phosphorylates km23-1 on serine 73 (S73). Moreover, our results show that the PKA-specific inhibitor H89 diminishes phosphorylation of km23-1 on S73 after TGFβ stimulation. Taken together, our results demonstrate that TGFβ induction of PKA activity results in phosphorylation of km23-1 on S73. In order to assess the mechanisms underlying PKA phosphorylation of km23-1 on S73 (S73-km23-1) after TGFβ stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFβ regulates complex formation between the PKA regulatory subunit RIβ and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGFβ induction of the km23-1-dynein complex and transcriptional activation of the activin-responsive element (ARE). Furthermore, our results show that km23-1 is required for cAMP-responsive element (CRE) transcriptional activation by TGFβ, with S73-km23-1 being required for the CRE-dependent TGFβ stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGFβ/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction.

Growth control of human ovarian cancer cells by the dynein light chain and TGFβ receptor interacting protein km23-1

We have identified km23-1 as a TGFβ signaling intermediate that is also a light chain of the minus-end directed motor protein dynein (DLC). Our results suggest that km23-1 (also termed DYNLRB1/robl1/mLC7-1/Dnlc2a) can function as a “motor receptor” to recruit TGFβ signaling components to the dynein motor for endosomal trafficking prior to nuclear translocation. For example, we have shown that overexpression of the dynactin subunit dynamitin, which is known to disrupt dynein-mediated intracellular transport, blocks TGFβ-stimulated nuclear translocation of Smad2. Further, siRNA blockade of km23-1 resulted in a reduction both in nuclear levels of phosphorylated (ie, activated) Smad2 and of TGFβ/Smad2-dependent transcriptional activity (JBC 282: 19122, 2007). Similarly, we have shown that km23-2 is required for Smad3 TGFβ signaling (JCP 221: 707, 2009). However, many human ovarian cancer cells (HOCCs) have lost growth inhibitory responsiveness to TGFβ. In order to assess whether km23-1 could also regulate the growth of aggressive, TGFβ-resistant HOCCs, we developed a tet-off-inducible model system for induction of wild-type km23-1 expression in SKOV-3 HOCCs. Here we show that overexpression of km23-1 (by 10-20 fold) suppressed the growth of the cells in vitro and markedly impeded tumor formation in vivo. Fluorescence-activated cell sorting (FACS) analysis in the SKOV-3 model system demonstrated an increased fraction of cells in G2/M after induction of km23-1 expression (dox removal), compared with controls. Similar results were obtained for the TGFβ-resistant IGROV-1 HOCCs. Additional studies revealed that km23-1 overexpression in HOCCs increased the mitotic index, in association with activation of both MEK and ERK and an elevation of cyclin B1 protein expression levels. The elevated levels of phospho-MEK, phospho-ERK, and cyclin B1, as well as the distribution of the cells in mitosis, suggest that the cells are delayed in prometaphase/ metaphase. This mitotic delay, followed by cell death, appears to be the mechanism by which high levels of km23-1 can suppress ovary tumor growth in vivo. Since dynein is known to have critical functions at this point in mitosis by inactivating the spindle assembly checkpoint (SAC), studies are underway to investigate how km23-1 overexpression regulates the SAC to cause the reduced tumor formation in vivo. This work was supported by NIH grants CA100239, CA92889, and CA90765 to KMM. Our results indicate that km23-1 is a critical ovarian cancer target for the development of novel diagnostic and therapeutic agents. In this regard, US patents have been issued to KMM related to km23-1-based methods for diagnostic/prognostic screening, as well as for the identification of km23-1-based therapeutic agents for ovarian cancer (US 7,229,758 B2; 6-07 & US 7,741,043; 6-10).