Phosphatidylinositol 3-Kinase C2α Contains a Nuclear Localization Sequence and Associates with Nuclear Speckles

Svetlana A Didichenko,Marcus Thelen

doi:10.1074/jbc.m104610200

Abstract

Phosphoinositide 3-kinase C2alpha (PI3K-C2alpha) belongs to the class II phosphatidylinositol 3-kinases, which are defined by their in vitro usage of phosphatidylinositol and phosphatidylinositol 4-phosphate as substrates. All type II phosphatidylinositol 3-kinases contain at their C terminus a C2-like domain. Here we demonstrate that Homo sapiens phosphoinositide 3-kinase C2alpha (HsPI3K-C2alpha) has dual cellular localization present in the cytoplasm and in the nucleus. A distinct nuclear localization signal sequence was identified by expressing HsPI3K-C2alpha-green fluorescent protein fusion proteins in HeLa cells. The nuclear localization signal was mapped to a stretch of 11 amino acids (KRKTKISRKTR) located within C2-like domain of the kinase. In the cytoplasm and the nucleus HsPI3K-C2alpha associates with macromolecular complexes that are resistant to detergent extraction. Indirect immunofluorescence reveals that in the nucleus HsPI3K-C2alpha is enriched at distinct subnuclear domains known as nuclear speckles, which contain pre-mRNA processing factors and are functionally connected to RNA metabolism. Phosphorylation of HsPI3K-C2alpha is induced by inhibition of RNA polymerase II-dependent transcription and coincides with enlargement and rounding up of the nuclear speckles. The results suggest that phosphorylation of HsPI3K-C2alpha is inversely linked to mRNA transcription and supports the importance of phosphoinositides for nuclear activity.

Highlights

Phosphatidylinositol 3-kinases (PI 3-kinases)1 have emerged as important constituents of cellular pathways regulating the remodeling of the cytoskeleton, the trafficking of intracellular organelles, and cell growth and survival [1]
The results suggest that phosphorylation of HsPI3K-C2␣ is inversely linked to mRNA transcription and supports the importance of phosphoinositides for nuclear activity
Nuclear and Cytoplasmic Localization of HsPI3K-C2␣ in Resting Cells—PI 3-kinases were shown to associate with different cellular compartments including the cytosol, the plasma membrane, endosomes, and the nucleus

Summary

EXPERIMENTAL PROCEDURES

Antibodies against PI3K-C2 were raised in rabbits immunized with chimeric proteins containing glutathione S-transferase fused either to amino acids 62–131 of murine p170 (GenBankTM accession number U55772) [27] or to the N terminus (amino acids 1–134) of HsPI3K-C2␣ (GenBankTM accession number Y13367) [28]. Subcellular Fractionation—HeLa cells (ϳ1–5 ϫ 107 cells) were treated with cytochalasin B (10 ␮g/ml) in culture medium for 30 min, trypsinized, washed 2 times with PBS, and resuspended in 1 ml of ice-cold hypotonic lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 10 ␮g/ml cytochalasin B, 40 mM NaF, 0.5 mM sodium orthovanadate, 40 mM ␤-glycerophosphate, 5 mM sodium pyrophosphate, and protease inhibitors (Complete, Roche Molecular Biochemicals)). Cells were washed twice in PBS and lysed in buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 1 mM EDTA) supplemented with phosphatase inhibitors (40 mM NaF, 0.5 mM sodium orthovanadate, 40 mM ␤-glycerophosphate, 5 mM sodium pyrophosphate) and protease inhibitors (Complete, Roche Molecular Biochemicals). Coverslips were washed extensively with PBS and once with water and mounted in polyvinyl alcohol (Gelvatol, Sigma) supplemented with 1% 1,4diazabicyclo[2,2,2]octane (Sigma)

RESULTS

DISCUSSION

PKKKRKV RLSRKR