In trodu ction .
In view of the profound inhibition of the IFNy antiviral response by JaklA B,
the most obvious site o f the inhibition was thought to be the Jak/STAT pathway, and thus a thorough analysis of it was undertaken. IFNy binding leads to phosphorylation
of J a k l, Jak2, IFNG Rl and ST ATI: inhibition of any of these would affect the response to ligand binding. Jak2 is the essential kinase activity for IFNy signalling,
Jakl kinase activity being essential only for the antiviral response, not for ST ATI activation (18). The phosphorylation of the receptor is required for STATl docking and subsequent tyrosine phosphorylation, which is obligatory for dimérisation and translocation to the nucleus for transcriptional regulation. There was no detectable inhibition by JaklAB o f receptor complex activation or STATl activation, nor was any effect detectable on the serine phosphorylation of STATl which is required for full transcriptional activity. Proteins thought to regulate the serine phosphorylation pathway itself were also unaffected. Importantly, it was discovered that JaklAB was constitutively phosphorylated, presumably reflecting significant (auto)kinase activity. The introduction of a kinase-inactivating mutation into JaklAB disrupted this constitutive phosphorylation and resulted in the loss of the inhibitory phenotype. Analysis of proteins by cellular fractionation was consistent with JaklAB being cytosolic and having no association with the receptor complex. This was consistent
Chapter 4. JaklAB has no detectable effect on the Jak/STAT pathway. 107
with there being no dominant negative effect on the Jak/STAT pathway and no ligand- dependent variation in the level of JaklAB phosphorylation.
R e su lts.
The Jak/STAT pathway is unaffected by JaklA B .
Receptor events and the activation by IFNy of the Jak/STAT 1 pathway in the
presence of JaklAB were analysed by immunoprécipitation and anti-phosphotyrosine Western blot analysis. In the 2fJaklAB clone #5 cells analysed - assuming comparable antibody specificity - the mutant Jak is expressed to slightly higher levels than the endogenous Jakl (Figure 3 .8 ). Tyrosine phosphorylation of the IFNy receptor 1
chain (IFN G R l), Ja k l, Jak2 and STATl in response to IFNy are unaffected by the
presence of JaklAB (Figure 4 .1 ). However, the mutant JaklAB is constitutively phosphorylated - in contrast to the inducible phosphorylation of wild type Jakl - which may have implications for the mechanism of the inhibition.
Consistent with the absence of an effect on tyrosine phosphorylation of the Jaks or ST A Tl, EMSA analysis showed no difference between wild-type and JaklAB- expressing cells in terms of the activation of STATl in response to IFNy or IF N a
(F igure 4 .2 ). The activation of STATl and STAT3 by Oncostatin M was also
unaffected (Figure 4.2).
Tyrosine phosphorylation is the essential step for ST AT activation, but the transcriptional activity of several STATs is also modulated through phosphorylation of conserved serine residues. In the case of STA Tl, phosphorylation of serine residue 727 is required for maximal transcriptional activity. There is increasing evidence that p38
Chapter 4. JaklAB has no detectable effect on the Jak/STAT pathway._______________ 108
MAP kinase (p38MAPK) can phosphorylate serine 727 and that it is activated in response to both a /p IFNs. More recently, IFNy was shown to activate p38 MAPK
and induce serine 727 phosphorylation (68), but there is conflicting evidence that it is p38 MAPK which is responsible for serine phosphorylation in response to IFNy (48).
Western blot analysis of the serine phosphorylation of STATl in 2fTGH and 2fJaklAB cells shows an increase following IFNy treatment (F ig u re 4 .3 ). No
inducible phosphorylation of p38 MAPK in response to IFNy was observed in either
cell line. Anisomycin - a potent activator of MAP kinases - did induce a strong auto phosphorylation of p38 MAPK, with corresponding phosphorylation of serine 727 in both cell lines (F ig u re 4 .3 ). JaklAB had no obvious effect on this serine 727 phosphorylation or that observed in response to IFNy (F ig u re 4 .3 ). The same blot
was reprobed, confirming the absence of any inhibition of the phosphorylation of tyrosine 701. Thus, JaklAB appears to be without effect on either tyrosine or serine phosphorylation of STATl in response to IFNy, and the observed activation of p38
MAPK.
In d u c tio n a n d a ctiv atio n o f d o u b le -stra n d e d R N A -d ep en d en t p ro te in k in a se (P K R ) is n o t in h ib ite d by Ja k lA B .
Prolonged IFN treatment leads to induction of PKR, which is involved in the response to both o /p IFNs as well as IFNy (118, 264). The autokinase activity of PKR
immunoprecipitated from IFN-treated cell extracts was assayed. A low-level increase in activity in wild-type cells accompanied treatment with IFNy, and this was not affected
by JaklAB (F ig u re 4 .4 c ). The inducible expression of PKR protein was also unaffected (F ig u re 4.4b).
Chapter 4. JaklAB has no detectable effect on the Jak/STAT pathway. 109
The inhibitory effect o f JaklA B is dependent on a functional kinase d o m a in .
Western blotting repeatedly showed significant constitutive tyrosine phosphorylation of JaklAB. It is recognised that overexpression of the Jak family kinases can lead to their autophosphorylation. For JaklAB it was possible that the resultant kinase-activity could be a major factor in the observed dominant negative effects of the mutant. To address this issue, a kinase negative JaklAB was generated using the inactivating lysine to glutamic acid substitution in motif II of the kinase domain (Hanks et a i, 1988). This new mutant was designated JaklABK>E.
Stable clones of 2fTGH cells expressing levels of JaklABK>E comparable to that of JaklAB were isolated. No apparent inhibitory effect of the JaklABK>E on the induction of CIITA or Class II mRNAs was seen (Figure 4 .5 ), nor on the antiviral response (da.a not presented). FACS analysis indicated a residual partial inhibition of the expression of Class II proteins in response to IFNy, but expression levels of
JaklABK>E did not seem to correlate with the extent of this residual inhibition (data not shown).
The cellular localisation o f JaklA B is different to endogenous J a k l.
The fact that the phosphorylation of JaklAB was not induced by IFNy, but seen
constitutively, suggested that the phosphorylation was not a result of interactions with the activated receptor complex. This possibility required verification because it would provide an insight into the site of the inhibitory effect. The subcellular distribution of JaklAB was investigated by preparing membrane and cytosolic protein fractions from 2fJaklAB cells. The mutant protein is cytoplasmic whilst endogenous Jakl is found at the membrane, presumably in association with cytokine-receptor complexes (F igure
Chapter 4. JaklAB has no detectable effect on the Jak/STAT pathway. 110
contamination of the cytoplasmic extract with membrane protein (Figure 4 .6 ), and lactate dehydrogenase assays (Figure 4 .7 ) demonstrated negligible contamination of membrane-associated extracts with cytosolic protein.
Chapter 4. JaklAB has no detectable effect on the Jak/STAT pathway._______________ 111
F igu re 4.1.
A nalysis o f the tyrosine phosphorylation o f com ponents o f the Jak /S T A T l pathw ay in response to IFNy in 2fTGH and 2fJaklA B .
2fTGH and 2fJakl AB#5 cells were plated out and the next day treated with IFNy for 15
minutes. Cells were harvested along with untreated controls and lysed in Schindler buffer. The concentration of protein in the extracts was determined and equal amounts used to immunoprecipitate Jak l, ST A Tl, Jak2 and IFN G R l. The immunoprecipitated proteins were resolved on a 6.5% SDS-PAGE gel and transfered to PVDF. The membrane was probed with anti phosphotyrosine antibodies, stripped and reprobed for Jak l, Jak2, STATl and IFNGRl protein, stripping after each Western blot.