Mutational analysis reveals an essential role for the LXXLL motif in the transformation function of the human herpesvirus-8 oncoprotein, kaposin.
Human herpesvirus-8 (HHV-8) is causally linked to Kaposi's sarcoma (KS). Sequence analysis of the genome and subsequent studies revealed several genes including kaposin, with transformation properties in cell culture. In this study, we have analyzed the requirement of Kaposin A for cellular transformation in an effort to understand its contribution towards KS pathogenesis. Comparative analysis of Kaposin with other proteins identified the LXXLL motif spanning from residues 31-35 (LVCLL). The observation that the LXXLL motif is present in nuclear receptor coactivators that mediate the interaction of coactivators with nuclear receptors has prompted us to investigate the relevance of this motif for Kaposin's function(s). Kaposin A coding sequences were cloned into a eukaryotic expression plasmid with the Flag (FL) epitope fused in-frame at the C-terminus (Kap-FL). To evaluate the role of leucine residues in the motif, site-directed mutagenesis was utilized, whereby alanine was substituted for the leucine residues (Kap-AXXAA-FL). Both Kap-FL and Kap- AXXAA-FL exhibited similar levels of expression in cells. Interestingly, the Kap-AXXAA-FL mutant failed to show transforming activity by two independent assays: anchorage-independent growth, and focus formation. Immunofluorescence (IFA) and FACS analysis indicated that Kap-FL was localized around the nucleus and at the cell surface, respectively. However, Kap-AXXAA-FL exhibited diffuse cytoplasmic staining as measured by IFA yet was still detectable on the cell surface by FACS. Ironically, both Kap-FL and Kap-AXXAAFL were able to activate the AP-1 promoter. These results support an important role for the LXXLL motif in the ability of Kaposin to induce transformation.
Tomkowicz
Brian
B
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Singh
Satya P
SP
Lai
Derhsing
D
Singh
Anjali
A
Mahalingham
Sundarasamy
S
Joseph
Jeymohan
J
Srivastava
Shiv
S
Srinivasan
Alagarsamy
A
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000276:immunology; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002472:Cell Transformation, Viral / Q000235:genetics; D004252:DNA Mutational Analysis; D019288:Herpesvirus 8, Human / Q000235:genetics; D006801:Humans; D007930:Leucine / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D012514:Sarcoma, Kaposi / Q000235:genetics / Q000378:metabolism / Q000821:virology; D018808:Transcription Factor AP-1 / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D009856:Oncogene Proteins, Viral; D018808:Transcription Factor AP-1; D014764:Viral Proteins; C120136:kaposin B protein, Human herpesvirus 8; D007930:Leucine