Twinfilin/A6
Kinase Classification: Atypical Group: Likely Non-Kinases
Twinfilin is an actin-binding protein that was once characterized as having protein kinase activity, and included in the human kinome catalog and those of several other species. However, it now appears as though the kinase activity report can not be replicated, and it is generally believed not to be a kinase. Twinfilin/A6 is found in most or all eukaryotes. It has two cofilin domains and is known to bind actin monomers. Human has a close paralog, A6r.
A6 was first cloned as a phospho-protein by Beeler et al [1], who isolated it with a phospho-tyrosine antibody in a bacterial expression screen. Since E. coli was not thought to have any tyrosine kinases, any tyrosine-phosphorylated protein was taken to be a tyrosine kinase. A beta-galactosidase-A6 fusion protein was produced in bacterial cells, immunoprecipitated and found to autophosphorylate in an in vito kinase activity; phosphoamino acid analysis showed phosphorylation on both tyrosine and threonine. The immunoprecipitate could also tyrosine phosphorylate MBP and poly-GluTyr, model kinase substrates. Kinase activity was Mn2+ or Mg2+ -dependent.
A6r was discovered by Rohwer et al[2] by two-hybrid interaction with PKC zeta; they show that both A6 and A6r bind ATP, however, they failed to see kinase activity by either protein. A second group [3] replicated the original Beeler in vitro kinase assay, using GST-A6 fusion protein purified by affinity chromatography and gel filtration, and were also unable to find any kinase activity. These two independent claims of lack of activity, coupled with a lack of homology to any known kinase, put against the single report of kinase activity caused us to remove the Twinfilin/A6 family from the kinome catalog.
What went wrong?
The original paper used reasonably convincing approaches to demonstrate kinase activity. How did they get it wrong? One possible error was in assuming that E. coli had no tyrosine kinase activity, since at least one tyrosine kinase, Wzc [4], has been characterized from coli. This may account for the tyrosine phosphorylation in the bacterial extract, and it is also possible that this or another kinase co-purified with A6 to give rise to the in vitro kinase activity, and that this contaminant did not appear in the studies by the two other groups. Whatever the case, this result casts some doubt on claims of kinase activity based only on immunopurified bacterial fusion proteins.
--Gerard 16:03, 15 April 2010 (PDT)
References
- Beeler JF, LaRochelle WJ, Chedid M, Tronick SR, and Aaronson SA. Prokaryotic expression cloning of a novel human tyrosine kinase. Mol Cell Biol. 1994 Feb;14(2):982-8. DOI:10.1128/mcb.14.2.982-988.1994 |
- Rohwer A, Kittstein W, Marks F, and Gschwendt M. Cloning, expression and characterization of an A6-related protein. Eur J Biochem. 1999 Jul;263(2):518-25. DOI:10.1046/j.1432-1327.1999.00537.x |
- Vincent C, Doublet P, Grangeasse C, Vaganay E, Cozzone AJ, and Duclos B. Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol. 1999 Jun;181(11):3472-7. DOI:10.1128/JB.181.11.3472-3477.1999 |
