The association of CaM and Hsp70 regulates S-phase arrest and apoptosis in a spatially and temporally dependent manner in human cells.
The cell cycle is controlled by regulators functioning at the right time and at the right place. We have found that calmodulin (CaM) has specific distribution patterns during different cell-cycle stages. Here, we identify cell-cycle-specific binding proteins of CaM and examine their function during cell-cycle progression. We first applied immunoprecipitation methods to isolate CaM-binding proteins from cell lysates obtained at different cell-cycle phases and then identified these proteins using mass spectrometry methods. A total of 41 proteins were identified including zinc finger proteins, ribosomal proteins, and heat shock proteins operating in a Ca(2+)-dependent or independent manner. Fifteen proteins were shown to interact with CaM in a cell-phase-specific manner. The association of the selected proteins and CaM were confirmed with in vitro immunoprecipitation and immunostaining methods. One of the identified proteins, heat shock protein 70 (Hsp70), was further studied with respect to its cell-cycle-related function. In vivo fluorescence resonance energy transfer (FRET) analysis showed that the interaction of CaM and Hsp70 was found in the nucleus during the S phase. Overexpression of Hsp70 is shown to arrest cells at S phase and, thus, induce cell apoptosis. When we disrupted the CaM-Hsp70 association with HSP70 truncation without the CaM-binding domain, we found that S-phase arrest and apoptosis could be rescued. The results suggest that the spatial and temporal association of CaM and Hsp70 can regulate cell-cycle progression and cell apoptosis.
Huang
Min
M
The Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, China.
Wei
Jun-Ning
JN
Peng
Wan-Xin
WX
Liang
Juan
J
Zhao
Chun
C
Qian
Yan
Y
Dai
Gu
G
Yuan
Jun
J
Pan
Fei-Yan
FY
Xue
Bin
B
Sha
Jia-Hao
JH
Li
Chao-Jun
CJ
D017209:Apoptosis; D002147:Calmodulin / Q000032:analysis / Q000378:metabolism*; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016196:S Phase
D002147:Calmodulin; D018840:HSP70 Heat-Shock Proteins