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Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways.

Jahnke M, Trowsdale J, Kelly AP - J. Biol. Chem. (2012)

Bottom Line: Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα.In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ.The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

ABSTRACT
HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

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Down-regulation of CD8-DMα reporter molecules by MARCH8. CD8-DM reporters were transiently expressed in HEK-293T cells together with EGFP-MARCH8, EGFP-MARCH8mut, or pEGFP. Cell surface expression of CD8 was measured by FACS using anti-CD8 mAb OKT8 and PE anti-mouse mAb (FL2). A, dot plots showing levels of CD8-DMα (upper) and CD8-DMα-K230R (lower) in cells co-expressing EGFP-MARCH8, EGFP-MARCH8 mutant, or pEGFP. B, histograms showing surface levels of CD8-DMα (left) and CD8-DMα-K230R (right) in cells expressing EGFP-MARCH8, as determined by EGFP expression using gates R4–6 of A. Data are representative of at least three independent experiments.
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Figure 1: Down-regulation of CD8-DMα reporter molecules by MARCH8. CD8-DM reporters were transiently expressed in HEK-293T cells together with EGFP-MARCH8, EGFP-MARCH8mut, or pEGFP. Cell surface expression of CD8 was measured by FACS using anti-CD8 mAb OKT8 and PE anti-mouse mAb (FL2). A, dot plots showing levels of CD8-DMα (upper) and CD8-DMα-K230R (lower) in cells co-expressing EGFP-MARCH8, EGFP-MARCH8 mutant, or pEGFP. B, histograms showing surface levels of CD8-DMα (left) and CD8-DMα-K230R (right) in cells expressing EGFP-MARCH8, as determined by EGFP expression using gates R4–6 of A. Data are representative of at least three independent experiments.

Mentions: To investigate the ubiquitination of DM, we generated a reporter construct containing the CD8α extracellular domain and the DMα transmembrane and cytoplasmic tail regions, an approach used previously for the analysis of HLA-DR ubiquitination (34). The amino acid composition of this and the other constructs are given in Table 1. We determined whether this reporter molecule was subject to regulation by MARCH8 because members of this family of E3 ligases are known to ubiquitinate classical class II molecules (22, 28, 29). Upon transient transfection of 293T cells, catalytically active MARCH8 was able to down-regulate CD8-DMα, whereas a catalytically inactive MARCH8 mutant did not (Fig. 1).


Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways.

Jahnke M, Trowsdale J, Kelly AP - J. Biol. Chem. (2012)

Down-regulation of CD8-DMα reporter molecules by MARCH8. CD8-DM reporters were transiently expressed in HEK-293T cells together with EGFP-MARCH8, EGFP-MARCH8mut, or pEGFP. Cell surface expression of CD8 was measured by FACS using anti-CD8 mAb OKT8 and PE anti-mouse mAb (FL2). A, dot plots showing levels of CD8-DMα (upper) and CD8-DMα-K230R (lower) in cells co-expressing EGFP-MARCH8, EGFP-MARCH8 mutant, or pEGFP. B, histograms showing surface levels of CD8-DMα (left) and CD8-DMα-K230R (right) in cells expressing EGFP-MARCH8, as determined by EGFP expression using gates R4–6 of A. Data are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3293585&req=5

Figure 1: Down-regulation of CD8-DMα reporter molecules by MARCH8. CD8-DM reporters were transiently expressed in HEK-293T cells together with EGFP-MARCH8, EGFP-MARCH8mut, or pEGFP. Cell surface expression of CD8 was measured by FACS using anti-CD8 mAb OKT8 and PE anti-mouse mAb (FL2). A, dot plots showing levels of CD8-DMα (upper) and CD8-DMα-K230R (lower) in cells co-expressing EGFP-MARCH8, EGFP-MARCH8 mutant, or pEGFP. B, histograms showing surface levels of CD8-DMα (left) and CD8-DMα-K230R (right) in cells expressing EGFP-MARCH8, as determined by EGFP expression using gates R4–6 of A. Data are representative of at least three independent experiments.
Mentions: To investigate the ubiquitination of DM, we generated a reporter construct containing the CD8α extracellular domain and the DMα transmembrane and cytoplasmic tail regions, an approach used previously for the analysis of HLA-DR ubiquitination (34). The amino acid composition of this and the other constructs are given in Table 1. We determined whether this reporter molecule was subject to regulation by MARCH8 because members of this family of E3 ligases are known to ubiquitinate classical class II molecules (22, 28, 29). Upon transient transfection of 293T cells, catalytically active MARCH8 was able to down-regulate CD8-DMα, whereas a catalytically inactive MARCH8 mutant did not (Fig. 1).

Bottom Line: Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα.In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ.The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

ABSTRACT
HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.

Show MeSH
Related in: MedlinePlus