Nucleolar targeting of the chaperone hsc70 is regulated by stress, cell signaling, and a composite targeting signal which is controlled by autoinhibition.
Hsc70s are constitutively synthesized members of the 70-kDa chaperone family; they are essential for viability and conserved among all organisms. When eukaryotic cells recover from stress, hsc70s accumulate in nucleoli by an unknown mechanism. Our studies were undertaken to characterize the signaling events and the targeting sequence required to concentrate hsc70 in the nucleoli of human cells. Here, we show that pharmacological inhibitors of phosphatidylinositol (PI) 3-kinase and MEK kinases as well as protein-tyrosine phosphatases abolished the stress-dependent nucleolar accumulation of hsc70. Furthermore, to identify the hsc70 nucleolar targeting sequence, green fluorescent protein-tagged fusion proteins with defined segments of hsc70 were generated and their subcellular distribution was analyzed in growing cells. These studies demonstrated that residues 225 to 297 serve as a heat-inducible nucleolar targeting signal. This segment directs green fluorescent protein to nucleoli in response to stress, but fails to do so under nonstress conditions. Fine mapping of the nucleolar targeting signal revealed that it has two separable functions. First, residues 225 to 262 direct reporter proteins constitutively to nucleoli, even without stress. Second, segment 263 to 287 functions as an autoinhibitory element that prevents hsc70 from concentrating in nucleoli when cells are not stressed. Taken together, PI 3-kinase and MEK kinase signaling as well as tyrosine dephosphorylation are essential for the accumulation of hsc70 in nucleoli of stressed cells. This process relies on a stress-dependent composite targeting signal that combines multiple functions.
Bański
Piotr
P
Department of Physiology, McGill University, Montreal H3G 1Y6, Canada.
Mahboubi
Hicham
H
Kodiha
Mohamed
M
Shrivastava
Sanhita
S
Kanagaratham
Cynthia
C
Stochaj
Ursula
U
D021581:Active Transport, Cell Nucleus; D002466:Cell Nucleolus / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D018833:Chaperonins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1 / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000378:metabolism*; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods
D050883:HSC70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D048728:MAP Kinase Kinase Kinase 1; D017027:Protein Tyrosine Phosphatases; D018833:Chaperonins
J Cell Sci. 2005 Apr 1;118(Pt 7):1335-7
15788650
Am J Physiol Cell Physiol. 2005 Oct;289(4):C1034-41
15930140
EMBO J. 1984 Dec 20;3(13):3095-100
6441707
Nucleic Acids Res. 2009 Jan;37(Database issue):D181-4
18984612
Cell Cycle. 2005 Aug;4(8):1036-8
16205120
Nature. 1990 Aug 16;346(6285):623-8
2143562
FEBS Lett. 2007 Jul 31;581(19):3702-10
17544402
Biotechniques. 1999 May;26(5):858-62
10337476
Am J Physiol Cell Physiol. 2007 Nov;293(5):C1427-36
17728396
Genes Dev. 2008 Jun 1;22(11):1427-38
18519635
FASEB J. 2001 Jun;15(8):1478-80
11387263
Histochem Cell Biol. 2005 Mar;123(3):203-16
15742198
J Gerontol A Biol Sci Med Sci. 2009 Feb;64(2):167-70
19228787
Mol Cell Proteomics. 2010 Jan;9(1):117-30
19812395
Mol Cell. 2005 Nov 23;20(4):513-24
16307916
BMC Mol Biol. 2007 Jan 25;8:6
17254328
N Engl J Med. 2005 Oct 6;353(14):1489-501
16207851
BMC Bioinformatics. 2007 Aug 29;8:316
17727718
Nat Rev Mol Cell Biol. 2007 Jul;8(7):574-85
17519961
Nature. 2006 May 25;441(7092):424-30
16724053
Biochemistry. 2008 Jul 8;47(27):7001-11
18557634
Oncogene. 2008 Oct 27;27(50):6489-506
18955975
J Biol Chem. 1989 Oct 25;264(30):18019-23
2553699
Biochem J. 2003 Dec 15;376(Pt 3):553-69
14531731
Sci Signal. 2008 Sep 16;1(37):pl2
18799422
Nat Rev Microbiol. 2007 Feb;5(2):119-27
17224921
Cell Mol Life Sci. 2008 Aug;65(15):2334-59
18408888
J Biol Chem. 2004 Mar 5;279(10):8867-72
14684748
Trends Biochem Sci. 2010 Jul;35(7):361-7
20363631
J Cell Biol. 2009 Mar 23;184(6):771-6
19289796
Cell Death Differ. 2004 Aug;11(8):862-74
15088071
J Neurosurg Anesthesiol. 2004 Jan;16(1):53-61
14676570
Semin Cell Dev Biol. 2004 Feb;15(1):17-29
15036203
Histochem Cell Biol. 2008 Jan;129(1):13-31
18046571
Mol Biol Cell. 2007 Oct;18(10):3966-77
17652456
Genes Dev. 2005 Mar 1;19(5):570-82
15741319
Cell Res. 2006 Jun;16(6):530-8
16775624
FEBS Lett. 2009 Jun 18;583(12):1987-93
19446553
Cell Mol Life Sci. 2008 Jan;65(1):113-27
17952368
Biochim Biophys Acta. 2007 Aug;1773(8):1213-26
17112607
Chromosoma. 2009 Feb;118(1):11-23
18925405
Nature. 2005 Jan 6;433(7021):77-83
15635413