Figure 1.

Expression and MT regulatory properties of nonphosphorylatable Ala- and Glu-substituted Op18 mutants. K562 cells were transfected with 12 μg DNA of each of the indicated pMEP4 derivatives, and hygromycin resistant cell lines were selected and induced with Cd ²⁺ (0.05 μM), as described in MATERIALS AND METHODS. (A) Transfected cell lines were Cd²⁺ induced for 24 h and phosphoisomers of Op18 were resolved by native PAGE and revealed using antibodies toward the C-terminus of Op18 (up to four Ser sites, Ser-16, Ser-25, Ser-38 and Ser-63, are phosphorylated in vivo). The faint band, at the position of Op18-tetraA that is observed in the lane depicting migration of Op18-tetraE, represents endogenous Op18. (B) Op18 levels were determined after 6 and 24 h of Cd²⁺ induction and data are presented as fold induction over endogenous Op18 (the endogenous Op18 level in K562 cells is ∼ 10 μM). (C) The fraction of polymerized tubulin in cells treated as under (B). Panels B and C show the mean of two independent transfection experiments.