FIGURE 3.

Lac1 and Lag1 are direct targets of Cka2. A, protein extracts from WT and cka2Δ cells expressing HA₃-tagged versions of Lac1 (PLY865 and PLY1044) and Lag1 (PLY890 and PLY1045) were prepared and resolved by SDS-PAGE and immunoblotted with α-HA antibody. B, immunopurified extracts from microsomal membrane fractions of WT cells expressing HA₃-tagged versions of Lac1 (PLY865) and Lag1 (PLY890) were treated with calf intestinal phosphatase (CIP) where noted, as described under “Experimental Procedures.” Samples bound and unbound (UB) to beads were resolved by SDS-PAGE and immunoblotted with α-HA antibody. C, N-terminal and C-terminal amino acid sequence of Lac1 and Lag1. The consensus CK2 sequence is in boldface type, with proposed phosphoserines underlined. The C-terminal dilysine ER retrieval motif is also noted with an underline. D, strain PLY1225 (lac1Δlag1Δ + pPL276 (WT Lac1)) was transformed with plasmids expressing WT Lag1 or various potential Lag1 CK2 phosphorylation site mutants, and strain PLY1226 (lac1Δlag1Δ + pPL277 (WT Lag1)) was transformed with plasmids expressing WT Lac1 or various potential Lac1 CK2 phosphorylation site mutants. The resulting transformants were streaked onto SCD minus uracil and leucine or onto 5-fluoroorotic acid (5-FOA) solid agar plates and grown at 30 °C for ∼2 days. E, protein extracts from WT and cka2Δ cells expressing HA₃-tagged versions of WT Lac1, WT Lag1, or various potential Lac1 or Lag1 CK2 phosphorylation site mutants were prepared and resolved by SDS-PAGE and immunoblotted with α-HA antibody. F, CK2 phosphorylation of recombinant GST-tagged C-terminal cytoplasmic portion of WT Lac1 and Lac1 serine to alanine (3S to A) mutant or GST alone. The [γ-³²P]ATP-labeled proteins were resolved by SDS-PAGE and quantified following autoradiography (P.I.= PhosphorImager). Averages are presented as mean values ± S.D. (error bars) (n = 3).