J. Larkin et al., Differential properties of two putative nuclear localization sequences found in the carboxyl-terminus of human IFN-gamma, J INTERF CY, 21(6), 2001, pp. 341-348
Interferon-gamma (IFN-gamma), a protein that uses the Jak-Stat pathway for
signal transduction, translocates rapidly to the nucleus in cells treated e
xtracellularly with the cytokine. A nuclear localization sequence (NLS) has
been identified and characterized in the C-terminus of IFN-gamma. Both hum
an and murine IFN-gamma contain this NLS. We show in this report that human
IFN-gamma (HuIFN-gamma) contains a second NLS at an upstream site, as dete
rmined in standard import assays using digitonin-permeabilized HeLa cells.
The primary sequence, analogous with the NLS sequence identified in murine
IFN-gamma, representing amino acids 122-132 of HuIFN-gamma was capable of m
ediating the nuclear import of the autofluorescent protein allophycocyanin
(APC) in an energy-dependent manner. The second sequence, representing amin
o acids 78-92 of HuIFN-gamma, was also capable of mediating the nuclear imp
ort of APC in an energy dependent manner but to a greatly reduced extent. T
he nuclear import of both sequences conjugated to APC was strongly blocked
by competition with unconjugated HuIFN-gamma (122-132). Competition by the
sequence HuIFN-gamma (78-92) effectively blocked the import of APC-conjugat
ed HuIFN-gamma (78-92) but, at the same concentration, was not capable of i
nhibiting the nuclear import of APC-conjugated HuIFN-gamma (122-132), sugge
sting that HuIFN-gamma (78-92) was a less efficient NLS than HuIFN-gamma (1
22-132). This is consistent with >90% loss of antiviral activity of HuIFN-g
amma lacking the downstream NLS in 122-132. The nuclear import of APC-conju
gated HLRIFN-gamma (122-132) was inhibited by a peptide containing the prot
otypical polybasic NLS of the SV40 T NLS, which suggests that the same Ran/
importin cellular machinery is used in both cases.