A deregulated activity of PKB/Akt (where PKB stands for protein kinase B) renders tumour cells resistant to a variety of apoptosis-inducing stimuli. Elucidation of the mechanisms responsible for this deregulation is of prime importance for the development of novel anti-cancer drugs. Results of the present study demonstrate that the constitutive activity of PKB/Akt in B16BL6 melanoma cells depends on the integrity of cholesterol-enriched membrane microdomains, since the exposure of cells to cholesterol-depleting agents decreases the phosphorylation of this enzyme, with no change in its total protein level. Inhibitors of Hsp90 (heat-shock protein 90) decreased phosphorylation of PKB/Akt with a similar pattern. Dephosphorylation of the enzyme, as a consequence of raft disintegration, could be precluded by inhibition of serine/threonine (but not tyrosine) phosphatases. Our results imply that destabilization of lipid rafts seemingly affects the association of Hsp90 with the respective serine/threonine phosphatases, thereby increasing the accessibility to PKB/Akt to deactivating phosphatases. We have found recently that reconstituted expression of H-2K class I glycoproteins in class I-deficient B16BL6 cells also decreases the phosphorylation of PKB/Akt. Therefore it is possible that raft-associated regulation of this important enzyme involves both H-2K glycoproteins and Hsp90.

Introduction

Activation of PKB/Akt (where PKB stands for protein kinase B) is essential for many cellular processes, including proliferation and escape from apoptosis. Hence it is not surprising that a deregulated constitutive activation of this enzyme is characteristic of a variety of malignant tumours. In many cases, the latter phenomenon is associated with deficiency of a negative PKB/Akt regulator, i.e. the lipid phosphatase PTEN [1]. However, even in PTEN-deficient tumours, the activation of PKB/Akt could be modulated by altering the composition of cholesterol-enriched membrane microdomains (rafts) [2], which accommodate a selected set of cell-surface proteins, including those tightly associated with transmembrane signal transduction [3].

We have found recently [4] that the resistance of MHC (major histocompctibility complex) class I- and II-deficient B16BL6 melanoma cells to apoptosis-inducing stimuli is associated with high levels of constitutively phosphorylated PKB/Akt, a phenomenon abrogated by the re-expression of an MHC class I-encoding gene in these cells. Since MHC class I glycoproteins are known to reside in lipid rafts, we decided to examine the importance of rafts integrity for regulating PKB/Akt [5]. Since MHC class I glycoproteins (H-2K) were found by us and others to regulate the signals elicited by membrane-associated tyrosine kinase receptors (insulin receptor) [4,6], which are known to reside in rafts and to activate PKB/Akt, an assumption was raised that MHC class I glycoproteins may be involved in determining the stability of rafts. To test this assumption, we decided to examine the importance of integrity of rafts for the activity of PKB/Akt.

Materials and methods

MHC class I-deficient and expressing clones of B16BL6 melanoma have been described in [7]. Cells were grown in RPMI 1640 medium with 10% of serum and antibiotics (all reagents were purchased from Biological Industries, Kibbutz Beit Haemek, Israel). The experiments employing MCD (methyl β-cyclodextrin), novobiocin, okadaic acid and tautomycin [all purchased from Sigma (Rehovot, Israel), except for Tautomycin (Biomol, Plymouth Meeting, PA, U.S.A.)] were performed in serum-free RPMI 1640 medium with antibiotics. Anti-Akt and anti-p-Akt antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and anti-Hsp90 antibody (where Hsp stands for heat-shock protein 90) was from StressGen Biotechnologies (San Diego, CA, U.S.A.)

Results and discussion

To test our assumption, MHC class I-deficient B16BL6 cells were exposed to a cholesterol-depleting agent, MCD. After 2 h of incubation, MCD induced a marked dephosphorylation of PKB/Akt without affecting its total protein level (Figures 1A and 1B). Similar results were observed in tumour cell lines other than B16BL6 melanoma (results not shown). We suggested that destabilization of rafts produced by cholesterol depletion renders PKB/Akt accessible to down-regulating phosphatases. These phosphatases are unlikely to be tyrosine phosphatases, since sodium orthovanadate could not prevent MCD-induced dephosphorylation (Figure 1A). Two serine/threonine PPs (protein phosphatases), PP2A and PP1, are known to dephosphorylate PKB/Akt; therefore MCD was combined with preferential inhibitors of either PP2A or PP1 (okadaic acid and tautomycin respectively) [8]. Only high concentrations of okadaic acid precluded MCD-induced dephosphorylation of PKB/Akt (Figure 1B), whereas tautomycin had no effect at all (results not shown), implying that a phosphatase distinct from PP2A and PP1 is involved in the raft-dependent down-regulation of PKB/Akt activity. It has been demonstrated recently [9] that an important regulator of PKB/Akt, Hsp90, is associated with lipid rafts. We were unable to demonstrate direct interactions between Hsp90 and PKB/Akt by means of immunoprecipitation (Figure 2A). However, inhibition of Hsp90 by incubating cells in the presence of novobiocin for 3 h (Figure 2B) or longer (results not shown) produced dephosphorylation of the enzyme in a mode similar to that induced by MCD. This raises the intriguing possibility that Hsp90 is involved in the sequestration of an as yet unidentified phosphatase in raft microdomains unless cells become exposed to either MCD or novobiocin. As mentioned above, the constitutive phosphorylation of PKB/Akt in B16BL6 cells is abrogated by the reconstituted expression of MHC class I glycoproteins in these cells [4]. Numerous reports have provided evidence that the latter glycoproteins reside in lipid rafts and combine with membrane receptors and other molecules involved in signal-transduction processes [5]; these intermolecular associations were found to be essential for the proper activity of signalling molecules [10]. Therefore the presence or absence of MHC class I molecules could influence the location, stability and function of other membrane constituents, including those composing the lipid rafts. The exact relationship between MHC class I, Hsp90, PKB/Akt and phosphatases is currently under intensive investigation in our laboratory.

Cholesterol-depleting agent induces dephosphorylation of PKB/Akt in class I-deficient B16BL6-8 and B16BL6-9 cells

Figure 1
Cholesterol-depleting agent induces dephosphorylation of PKB/Akt in class I-deficient B16BL6-8 and B16BL6-9 cells

(A) The levels of phosphorylated (p-Akt) and total PKB/Akt (Akt) were monitored by immunoblotting in B16BL6-8 cells treated with 1% MCD combined with the indicated concentrations of sodium orthovanadate (NaVa). (B) The levels of phosphorylated (p-Akt) and total PKB/Akt (Akt) were monitored in B16BL6 cells treated with 1% MCD combined with the indicated concentrations of okadaic acid (OA).

Figure 1
Cholesterol-depleting agent induces dephosphorylation of PKB/Akt in class I-deficient B16BL6-8 and B16BL6-9 cells

(A) The levels of phosphorylated (p-Akt) and total PKB/Akt (Akt) were monitored by immunoblotting in B16BL6-8 cells treated with 1% MCD combined with the indicated concentrations of sodium orthovanadate (NaVa). (B) The levels of phosphorylated (p-Akt) and total PKB/Akt (Akt) were monitored in B16BL6 cells treated with 1% MCD combined with the indicated concentrations of okadaic acid (OA).

Inhibition of Hsp90 by novobiocin induces dephosphorylation of PKB/Akt in class I-deficient B16BL6 cells

Figure 2
Inhibition of Hsp90 by novobiocin induces dephosphorylation of PKB/Akt in class I-deficient B16BL6 cells

(A) PKB/Akt was precipitated using the appropriate antibodies and probed with either anti-Akt or anti-Hsp90 antibodies. (B) The levels of phosphorylated (p-Akt) and total PKB/Akt (Akt) were monitored by immunoblotting in B16BL6-8 and B16BL6-9 cells treated with 0.5 mM novobiocin (NB) for 3 h.

Figure 2
Inhibition of Hsp90 by novobiocin induces dephosphorylation of PKB/Akt in class I-deficient B16BL6 cells

(A) PKB/Akt was precipitated using the appropriate antibodies and probed with either anti-Akt or anti-Hsp90 antibodies. (B) The levels of phosphorylated (p-Akt) and total PKB/Akt (Akt) were monitored by immunoblotting in B16BL6-8 and B16BL6-9 cells treated with 0.5 mM novobiocin (NB) for 3 h.

Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).

Abbreviations

     
  • Hsp90

    heat-shock protein 90

  •  
  • MCD

    methyl β-cyclodextrin

  •  
  • MHC

    major histocompatibility complex

  •  
  • PKB

    protein kinase B

  •  
  • PP

    protein phosphatase

We thank Dr E. Gorelik (Department of Pathology, University of Pittsburgh, Pittsburgh, PA, U.S.A.) for providing us with the B16BL6 cell line.

References

References
1
Brader
S.
Eccles
S.A.
Tumori
2004
, vol. 
90
 (pg. 
2
-
8
)
2
Zhuang
L.
Lin
J.
Lu
M.L.
Solomon
K.R.
III
Freeman
M.
Cancer Res.
2002
, vol. 
62
 (pg. 
2227
-
2231
)
3
Brown
D.A.
London
E.
Annu. Rev. Cell Dev. Biol.
1998
, vol. 
14
 (pg. 
111
-
136
)
4
Assa-Kunik
E.
Fishman
D.
Kellman-Pressman
S.
Tsory
S.
Elhyany
S.
Baharir
O.
Segal
S.
J. Immunol.
2003
, vol. 
171
 (pg. 
2945
-
2952
)
5
Fishman
D.
Elhyany
S.
Segal
S.
Folia Biol. (Praha)
2004
, vol. 
50
 (pg. 
35
-
42
)
6
Stagsted
J.
APMIS Suppl.
1998
, vol. 
85
 (pg. 
1
-
40
)
7
Gorelik
E.
Kim
M.
Duty
L.
Henion
T.
Galili
U.
Clin. Exp. Metastasis
1993
, vol. 
11
 (pg. 
439
-
452
)
8
Resjo
S.
Goransson
O.
Harndahl
L.
Zolnierowicz
S.
Manganiello
V.
Degerman
E.
Cell Signal.
2002
, vol. 
14
 (pg. 
231
-
238
)
9
Sehgal
P.B.
Acta Biochim. Pol.
2003
, vol. 
50
 (pg. 
583
-
594
)
10
Damjanovich
S.
Matyus
L.
Damjanovich
L.
Bene
L.
Jenei
A.
Matko
J.
Gaspar
R.
Szollosi
J.
Immunol. Lett.
2002
, vol. 
82
 (pg. 
93
-
99
)