Polycystin-2 associates with tropomyosin-1, an actin microfilament component

Qiang Li, Yue Dai, Lei Guo, Yan Liu, Chunhai Hao, Guanqing Wu, Nuria Basora, Marek Michalak, Xing Zhen Chen

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel. PC2 has been shown to connect indirectly with the actin microfilament. Here, we report a direct association between PC2 and the actin microfilament. Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex. Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells. We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2. In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms. Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a. Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells. Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs. Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney. Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered.

Original languageEnglish (US)
Pages (from-to)949-962
Number of pages14
JournalJournal of Molecular Biology
Volume325
Issue number5
DOIs
StatePublished - Jan 31 2003
Externally publishedYes

Fingerprint

Tropomyosin
Actin Cytoskeleton
Protein Isoforms
Kidney
Immunoprecipitation
polycystic kidney disease 2 protein
Autosomal Dominant Polycystic Kidney
3T3 Cells
NIH 3T3 Cells
Alternative Splicing
Indirect Fluorescent Antibody Technique
Growth
Xenopus
Population Groups
Oocytes
Cations
Cysts
Proteins
Fibroblasts
Yeasts

Keywords

  • ADPKD
  • Co-immunoprecipitation
  • PKD2
  • TM-1
  • Yeast two-hybrid screen

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Polycystin-2 associates with tropomyosin-1, an actin microfilament component. / Li, Qiang; Dai, Yue; Guo, Lei; Liu, Yan; Hao, Chunhai; Wu, Guanqing; Basora, Nuria; Michalak, Marek; Chen, Xing Zhen.

In: Journal of Molecular Biology, Vol. 325, No. 5, 31.01.2003, p. 949-962.

Research output: Contribution to journalArticle

Li, Q, Dai, Y, Guo, L, Liu, Y, Hao, C, Wu, G, Basora, N, Michalak, M & Chen, XZ 2003, 'Polycystin-2 associates with tropomyosin-1, an actin microfilament component', Journal of Molecular Biology, vol. 325, no. 5, pp. 949-962. https://doi.org/10.1016/S0022-2836(02)01333-5
Li, Qiang ; Dai, Yue ; Guo, Lei ; Liu, Yan ; Hao, Chunhai ; Wu, Guanqing ; Basora, Nuria ; Michalak, Marek ; Chen, Xing Zhen. / Polycystin-2 associates with tropomyosin-1, an actin microfilament component. In: Journal of Molecular Biology. 2003 ; Vol. 325, No. 5. pp. 949-962.
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AU - Wu, Guanqing

AU - Basora, Nuria

AU - Michalak, Marek

AU - Chen, Xing Zhen

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AB - Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel. PC2 has been shown to connect indirectly with the actin microfilament. Here, we report a direct association between PC2 and the actin microfilament. Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex. Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells. We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2. In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms. Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a. Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells. Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs. Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney. Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered.

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