Mutant constructs used to study CKAP4 phosphorylation and palmitoylation. CKAP4 mutants that mimic constitutive depalmitoylation and various states of serine (S3, S17 and S19) phosphorylation were generated to determine their effect on the subcellular distribution of CKAP4 in response to APF. CKAP4SΔE translocated from the PM to the nucleus in response to APF; CKAP4SΔA localized to the PM but did not translocate to the nucleus in response to APF; none of the C100S mutants were expressed on the PM; CKAP4C100S/SΔE was expressed in the nucleus. The results indicate that CKAP4 must be palmitoylated for PM expression and depalmitoylated and phosphorylated for translocation to the nucleus. A summary of these results are provided in the table.
| Name||Mutations||Comments posttranslational modifications||Resting distribution||Distribution after APF|
|wild-type (WT) CKAP4||none||expressed or endogenous||PM, ER||> nucleus|
|CKAP4 C100S||C100S||constitutively depalmitoylated||ER||= ER|
|CKAP4 SΔA||S3A, S17A, S19A||constitutively dephosphorylated||PM, ER||= PM, ER|
|CKAP4 C100S/SΔA||C100S, S3A, S17A, S19A||constitutively depalmitoylated, constitutively dephosphorylated||ER||ER|
|CKAP4 SΔE||S3E, S17E, S19E||mimics phosphorylation||PM, ER||> nucleus|
|CKAP4 C100S/SΔE||C100S, S3E, S17E, S19E||constitutively depalmitoylated, mimics phosphorylation||Nucleus||= nucleus|