Mutants of the protein serine kinase PSKH1 disassemble the Golgi apparatus.

We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.

PSKH1, a novel splice factor compartment-associated serine kinase.

Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in splicing factor compartments (SFCs) within the nucleus of interphase cells. Nuclear SFCs are considered mainly as storage sites for splicing factors, supplying splicing factors to active genes. The mechanisms controlling the interaction of the various spliceosome constituents, and the dynamic nature of the SFCs, are still poorly understood. We show here that endogenous PSKH1, a previously cloned kinase, is located in SFCs. Migration of PSKH1-FLAG into SFCs is enhanced during co-expression of T7-tagged ASF/SF2 as well as other members of the SR protein family, but not by two other non-SR nuclear proteins serving as controls. Similar to the SR protein kinase family, overexpression of PSKH1 led to reorganization of co-expressed T7-SC35 and T7-ASF/SF2 into a more diffuse nuclear pattern. This redistribution was not dependent on PSKH1 kinase activity. Different from the SR protein kinases, the SFC-associating features of PSKH1 were located within its catalytic kinase domain and within its C-terminus. Although no direct interaction was observed between PSKH1 and any of the SR proteins tested in pull-down or yeast two-hybrid assays, forced expression of PSKH1-FLAG was shown to stimulate distal splicing of an E1A minigene in HeLa cells. Moreover, a GST-ASF/SF2 fusion was not phosphorylated by PSKH1, suggesting an indirect mechanism of action on SR proteins. Our data suggest a mutual relationship between PSKH1 and SR proteins, as they are able to target PSKH1 into SFCs, while forced PSKH1 expression modulates nuclear dynamics and the function of co-expressed splicing factors.