A synthetic peptide corresponding to residues 65–79 of the HLA class II allele DQA*03011 inhibits IL-2–mediated proliferation in a manner similar to that of rapamycin. DQ 65-79 does not affect the expression of the IL-2R or inhibit phosphorylation of the IL-2R β chain. Although both DQ 65-79 and rapamycin inhibit p70 S6 kinase activity, only DQ 65-79 also blocks Akt kinase activity. In addition, there is striking homology between residues 70–79 of DQ 65-79 and the p110 subunit of PI 3-kinase. Whereas DQ 65-79 has no effect on the association of the two subunits of PI 3-kinase, it directly inhibits PI 3-kinase activity in vitro. Furthermore, confocal microscopy of cells treated with a tagged form of DQ 65-79 shows that it rapidly appears in the cytosol, where it can associate with PI 3-kinase. Thus, DQ 65-79 blocks the IL-2R signal transduction pathway by inhibiting PI 3-kinase activity.
Our initial hypothesis was that DQ 65-79 physically interfered with the interaction between T cells and antigen-presenting cells by binding to the T-cell receptor. However, our findings do not support this. DQ 65-79 inhibits not only allorecognition but also proliferation of purified T cells in response to anti-CD3 or IL-2, indicating that antigen-presenting cells are not required for inhibition. Interestingly, the peptide does not inhibit PMA- and ionomycin-mediated proliferation (15
), implying a membrane-associated effect. Additionally, DQ 65-79 inhibits intracellular signaling events in a manner similar to but distinct from the immunosuppressive drugs rapamycin and wortmannin, suggesting that DQ 65-79 acts within the cell. The observation that DQ 65-79 is rapidly internalized in cells (Drouvalakis and Krensky, manuscript in preparation) supports an intracellular locus for peptide action. Thus, the functional activities and physical location of DQ 65-79 are consistent with inhibition of PI 3-kinase activity.
Although the mechanism by which DQ 65-79 crosses the membrane has not been established, other peptides also translocate into cells. For example, conjugation of proteins to short cationic peptides enhances transport of proteins into cells (51
). Zhang and coworkers prepared cell-permeable synthetic peptides by incorporation of a variety of hydrophobic sequences (53
). DQ 65-79 is mainly composed of hydrophobic and positively charged residues. Substitution analysis showed that replacement with serine at all but two of these residues resulted in a reduction of inhibitory activity (15
). Thus, it is possible that the ability of DQ 65-79 to translocate into the cytosol is related to its overall hydrophobicity and positively charged amino acids.
Murphy and coworkers recently described a peptide designated HLA-DQA1 that inhibits allorecognition by inducing apoptosis (54
). This peptide is derived from residues 62–77 of the α chain of DQA*0101, but differs from DQ 65-79 in several ways: (a) HLA-DQA1 is three residues longer than DQ 65-79 at the amino terminus; (b) HLA-DQA1 is two residues shorter than DQ 65-79 at the carboxyl terminus; and (c) the core region of the two peptides differs at residues 66, 69, and 76. It will be interesting to determine whether these two related peptides have similar mechanisms of action.
We have not yet determined exactly how DQ 65-79 interferes with PI 3-kinase activity. The region of homology between the peptide and p110 is within the catalytic region of the subunit that contains several functional subdomains (50
). The DQ 65-79 homology region does not correspond to the ATP-binding region (amino acids 802–825) (50
), to the covalent attachment site of wortmannin (amino acid 802) (55
), or to the putative head binding region (amino acids 937–952) (56
); rather, it corresponds to part of the kinase domain (amino acids 916–924) (50
). Interestingly, a change at position 916 of p110-β from arginine to lysine causes a complete loss of kinase function (57
). The corresponding residue in DQ 65-79 is lysine instead of arginine, which may account for the inhibitory effects of the peptide. However, we are unable to demonstrate direct interaction between DQ 65-79 and PI 3-kinase, ATP, or the lipid substrate (Boytim and Clayberger, unpublished observations). The interaction of the peptide with the components of the in vitro kinase assay may be too weak to be detected by conventional methods. Alternatively (though it is not likely), the peptide may be acting through an associated protein that is coimmunoprecipitated with the kinase.
Due to the ubiquitous expression of PI 3-kinase and its role in many different signaling pathways, one might expect that DQ 65-79 would inhibit multiple cell types and multiple effector functions of those cells. We have observed a range in sensitivity to the antiproliferative effects of the peptide with different cell types, with lymphocytes being more sensitive than other types of cells. This may be attributed to the levels of different isoforms of PI 3-kinase, although it is not yet clear what role the different isoforms play in signaling specificity. It is intriguing that the p110-δ isoform is primarily leukocyte specific (58
), which may provide an explanation for the more potent effects of DQ 65-79 on lymphocytes vs. other cell types.
DQ 65-79 may also inhibit the activity of other PI kinase family members. Wortmannin has been shown to block the kinase activity of PI 4-kinase (59
), mTOR (60
), and Vps34p (61
). The PI kinase family member mTOR also has homology to DQ 65-79 in its kinase domain. Although no lipid kinase activity has been demonstrated for mTOR (56
), the peptide may affect other activities of mTOR. Inhibition of multiple members of the IL-2R signaling pathway may explain the strong inhibitory effect of the peptide on proliferation, but the effect of the peptide on mTOR and other PI kinase family members remains to be investigated. It is also possible that the peptide may interact with other proteins within the cell. Therefore, although we have demonstrated the inhibition of PI 3-kinase as a primary action of DQ 65-79, other effects may also be involved in the inhibition of proliferation.