Carboxy terminus of polyoma middle-sized tumor antigen is required for attachment to membranes, associated protein kinase activities, and cell transformation.

Abstract

We have constructed a transformation-defective polyoma virus mutant (Py 1387-T) that directs the synthesis of a normal small tumor antigen, a functional large tumor antigen, and a truncated (51,000-dalton) middle-sized tumor (mT) antigen that lacks 37 amino acids at its COOH terminus. The shortened mT polypeptide is missing the hydrophobic "tail" thought to be responsible for the anchorage of this protein into the plasma membrane and is in fact in cytosol fractions. This truncated mT polypeptide is inactive in an in vitro protein kinase assay and is altered in its phosphorylation in viva Mutant 1387-T differs from wildtype virus in having a T-A base pair instead of a C-G base pair at nucleotide position 1387. This change was introduced into viral DNA by using a synthetic undecanucleotide as a specific mutagen. Wild-type polyoma DNA was rendered single stranded by molecular cloning into coliphage M13. The oligonucleotide, which hybridizes with a mismatch at the site to be altered, was used to prime the synthesis ofdouble-stranded closed circular DNA. Progeny recombinant phages were screened by DNA sequence analysis for the desired base change. The polyoma mutant was reconstructed from recombinant phage replicative form DNA molecules containing the mutation.The early region of polyoma virus encodes three proteins, the small [22-kilodalton (kDal)], the middle-sized (56-kDal), and the large (100-kDal) tumor (T) antigens. The large T antigen is required for transformation by intact virus (1-3) and may function by promoting stable association ofthe viral genome with cellularDNA. An intact large T protein is not essential for maintenance ofthe transformed phenotype (4)(5)(6)(7)(8)(9). Small T and middle T (mT) antigens have been implicated in maintenance of the transformed state and in tumorigenicity by the virus. The nontransforming hr-t mutants (10-12) have lesions affecting both the small T and mT polypeptides but not large T protein (4,(13)(14)(15).The importance of the mT polypeptide in transformation has been demonstrated directly; DNA coding only for mT antigen can transform rat cells (16). Mutants in which mT, but not small T, antigen is altered are affected in their transformation properties (17-19). When deletions affecting the COOH-terminal region of mT antigen are created in cloned polyoma DNA, the ability of that DNA to transform cells is reduced (20).Phosphorylation is important to mT antigen function. A protein kinase activity that phosphorylates tyrosine residue 315 of mT antigen is present in T-antigen immunoprecipitates (21-25). Middle T antigen can be resolved into two species, 56 kDal and 58 kDal, that differ strikingly in activity in the in vitro kinase reaction (24,25). These species also differ in their patterns of phosphorylation in vivo. The high specific activity 58-kDal form is phosphorylated in vivo at different serine or threonine residues than is the 56-kDal form (24, 25). These results suggest that the activity of mT antigen may be regulated by cellular protein kinase(s).mT antig...

Large-scale production of polyoma middle T antigen by using genetically engineered tumors.

“…It is a membranebound protein anchored by a stretch of hydrophobic amino acids at its C terminus (4,12,28). There are two forms of middle T, a predominant 56,000-molecular-weight (56K) species and a minor 58K species (23,24) differing in their serine and threonine phosphorylation.…”

“…Middle T antigen of polyomavirus is required for cell transformation (4,5,10,13,16,20,22,30). It is a membranebound protein anchored by a stretch of hydrophobic amino acids at its C terminus (4,12,28).…”

Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G.

“…The protein kinase phosphorylates a tyrosine residue of the MT antigen in immunoprecipitates (6). With few exceptions (18,24), mutations in the MT antigen that affect cell transformation also affect the protein kinase activity, suggesting that the activity may be involved in transformation (1,6,24,28,31). However, polyomavirustransformed cells do not have elevated levels of phosphotyrosine in total cellular protein, as do cells transformed by retroviruses encoding tyrosine-specific protein kinases (26).…”

“…Recombinant DNA plasmids encoding only the MT antigen can transform established cell lines (32). Many mutations affecting the MT antigen reduce or abolish cell transformation (1,5,11,15,(17)(18)(19)31). Although the MT antigen alone can transform established cell lines, a portion of the large T antigen is required in addition for transforming primary cells (20).…”

“…However, the hydrophobic region of the MT antigen appears to be important for membrane association, cell transformation, and protein kinase activity. MT antigens lacking the hydrophobic region fail to become associated with cell membranes, to transform cells, or to show the associated protein kinase activity (1,31).…”