Changes in cytoskeletal organization in polyoma middle T antigen-transformed fibroblasts: involvement of protein phosphatase 2A and src tyrosine kinases.
The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen-induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T(1/2) mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole-induced disassembly. These effects were associated with a modest but statistically significant (P</=0.05) increase in recovery of protein phosphatase 2A activity with microtubules. Middle T antigen expression also depleted the normal cellular complement of actin stress fibers and focal adhesions, in parallel with changes in the distribution of src tyrosine kinases. Herbimycin A promoted recovery of paxillin and phosphotyrosine into nascent focal adhesion sites, in addition to restoring normal src tyrosine kinase distribution. However, herbimycin A did not restore actin stress fibers or parental-type microtubules to Pym T cells. We suggest that regulation of the microtubule array by middle T antigen may occur through direct effects including redistribution of protein phosphatase 2A as well as indirect effects such as altered interactions with actin-based stress fibers.
da Costa
S R
SR
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90033, USA.
Wang
Y
Y
Vilalta
P M
PM
Schönthal
A H
AH
Hamm-Alvarez
S F
SF
D000199:Actins / Q000378:metabolism; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D016227:Benzoquinones; D002460:Cell Line; D002461:Cell Line, Transformed; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D047029:Lactams, Macrocyclic; D051379:Mice; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D051419:Paxillin; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D054648:Protein Phosphatase 2; D011809:Quinones / Q000494:pharmacology; D017828:Rifabutin / Q000031:analogs & derivatives; D022502:Stress Fibers / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism*
D000199:Actins; D000952:Antigens, Polyomavirus Transforming; D000970:Antineoplastic Agents; D016227:Benzoquinones; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D011809:Quinones; D014746:Vimentin; D017828:Rifabutin; D019000:Phosphotyrosine; D014443:Tyrosine; C020754:herbimycin; D019061:src-Family Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D015739:Nocodazole