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

Abstract

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.

Fig. 1. TAP of km23-1 interacting protein complexes

A. 293T cells were transiently transfected with either EV or km23-1-TAP, and protein complexes were purified by the TAP protocol described in the “Materials and Methods.” Aliquots were analyzed by Western blotting. All data are representative of three independent experiments. B. Protein complexes obtained as in A were resolved by 5–20% SDS-PAGE. The gel was visualized with Sypro Ruby Red stain. The indicated bands were excised from the gel and identified by MS. The results are representative of two independent experiments.

Fig. 2. Over-expression of km23-1 increases actin filaments in MDCK cells stably expressing km23-1

Actin immunostaining analyses were performed as described in “Materials and Methods,” and representative photos are shown (400X).

Fig. 3. KD of km23-1 decreases basal RhoA activation in Mv1Lu epithelial cells

Mv1Lu cells were transiently transfected with either NC siRNA or km23-1 siRNA. 24 h after transfection, RhoA activation assays were performed as described in “Materials and Methods.”

Fig. 4. Depletion of km23-1 inhibited cell migration of HCCCs

Wound-healing assays were performed as described in “Materials and Methods.” Photographs were taken immediately after wounding and 24 h later. The results were quantified and normalized to parental control cells. Data plotted are the mean ± SE of triplicate wells from a representative experiment (n = 4). Asterisk (*) indicates P < 0.01 compared with control cells. All data are representative of two independent experiments.