Two novel phosphatidylinositol-4-phosphate 5-kinase type Igamma splice variants expressed in human cells display distinctive cellular targeting

Abstract

The generation of various phosphoinositide messenger molecules at distinct locations within the cell is mediated via the specific targeting of different isoforms and splice variants of phosphoinositide kinases. The lipid messenger PtdIns(4,5)P(2) is generated by several of these enzymes when targeted to distinct cellular compartments. Several splice variants of the type Igamma isoform of PIPK (PtdIns4P 5-kinase), which generate PtdIns(4,5)P(2), have been identified, and each splice variant is thought to serve a unique functional role within cells. Here, we have identified two novel C-terminal splice variants of PIPKIgamma in human cells consisting of 700 and 707 amino acids. These two splice variants are expressed in multiple tissue types and display PIPK activity in vitro. Interestingly, both of these novel splice variants display distinct subcellular targeting. With the addition of these two new splice isoforms, there are minimally five PIPKIgamma splice variants that have been identified in mammals. Therefore, we propose the use of the HUGO (Human Genome Organization) nomenclature in the naming of the splice isoforms. PIPKIgamma_i4 (700 amino acids) is present in the nucleus, a targeting pattern that has not been previously observed in any PIPKIgamma splice variant. PIPKIgamma_i5 (707 amino acids) is targeted to intracellular vesicle-like structures, where it co-localizes with markers of several types of endosomal compartments. As occurs with other PIPKIgamma splice variants, the distinctive C-terminal sequences of PIPKIgamma_i4 and PIPKIgamma_i5 may facilitate association with unique protein targeting factors, thereby localizing the kinases to their appropriate cellular subdomains for the site-specific generation of PtdIns(4,5)P(2).

Figure 1. The human PIPKIγ gene encodes at least four C-terminal splice variants

(A) The full-length PIPKIγ_v4 and PIPKIγ_v5 transcripts were amplified from MCF10A-epithelial-cell cDNA using a primer targeted to the conserved 5′-end of the PIPKIγ transcript and a second primer matching the putative 3′ alternatively spliced exon identified via 3′-RACE. The less prominent band running immediately below PIPKIγ_v5 was also sequenced and found to be non-specific. (B) A revised exon map of the human PIPKIγ gene illustrates the four major C-terminal splice variants that have been identified. The overall lengths of PIPKIγ transcripts listed are approximate estimates based on the defined open reading frames plus the 3′ untranslated region. Two novel exons, which have been termed exon 16b and 16c, make up the alternatively spliced C-termini of the PIPKIγ_v4 and PIPKIγ_v5 transcripts. Interestingly, the PIPKIγ_v4 transcript is longer than that of PIPKIγ_v5, but PIPKIγ_v4 mRNA encodes a shorter protein, owing to a stop codon in exon 16b. PIPKIγ transcripts were identified in our initial experiments that lacked portions of exons 3 or 5, or all of exon 14, but these splice variants were not further characterized. (C) CLUSTALW alignment of the C-terminal amino acid residues of the four PIPKIγ splice variants. Note that part of the C-terminus of PIPKIγ_i5 (W⁶⁴⁷IYSPRH⁶⁵³) is similar to the C-terminus of PIPKIγ_i2 (W⁶⁴⁷VYSPLH⁶⁵³). The presence of a full stop (period) indicates the lack of a corresponding amino acid residue at the indicated position.

Figure 2. PIPKIγ_i4 and PIPKIγ_i5 are evolutionarily conserved

A CLUSTALW alignment of putative orthologues of PIPKIγ_i4 and PIPKIγ_i5 was created using sequence information in the ENSEMBL database. The C-terminal amino acid sequences of PIPKIγ_i4 and PIPKIγ_i5 were used to search the ENSEMBL sequence database for potential matches in other species. Key to species not already identified: C. familiaris, Canis familiaris (dog); G. gallus, Gallus gallus (chicken); H. sapiens, Homo sapiens (man); M. musculus, Mus musculus, house mouse; R. norvegicus, Rattus norvegicus, Norway rat.

Figure 3. PIPKIγ_v4 and PIPKIγ_v5 transcripts are expressed as proteins

(A) The specificity of purified polyclonal antibodies toward the unique C-terminal splice variants of PIPKIγ was tested via Western blot. HA-tagged PIPKIγ constructs were transfected into HeLa cells, and Western blots of whole-cell lysates were probed with splice variant-specific anti-PIPKIγ polyclonal antibodies. (B) To verify that the anti-PIPKIγ polyclonal antibodies can recognize endogenous protein and are specific towards their intended splice variant, total PIPKIγ was knocked down in HeLa cells using siRNA for 48 or 72 h. Cell lysates were Western-blotted using each of the anti-PIPKIγ polyclonal antibodies, and anti-actin antibody was used as a loading control. (C) Expression of PIPKIγ splice variants in several mammalian cell lines was determined by Western-blotting cell line lysates with anti-PIPKIγ antibodies. Anti-actin antibody was used as a loading control. (D) Fresh tissue was extracted from a C57BL/6 mouse, lysed, and total soluble protein was quantified. A 20 μg portion of lysate was subjected to Western blotting with the anti-PIPKIγ polyclonal antibodies to determine the tissue distribution of splice variants. Abbreviations: Ctrl., control; HA-Iγ, HA-tagged PIPKIγ; IB:, immunoblot; Iγ_i5 (etc.), PIPKIγ_i5; Lrg. Int., lareg intestine; pan-Iγ, pan-PIPKIγ; Transfect., transfection. i_5 etc. designates the protein, whereas v_5 designates the mRNA.

Figure 4. PIPKIγ splice variants display PtdIns(4)P 5-kinase activity

Either 1 μg (1X) or 5 μg (5X) of His₆-tagged recombinant PIPKIα or PIPKIγ splice variants were added to PtdIns4P micelles and [γ-³²P]ATP for 5 min at room temperature to test in vitro PIPK activity, and lipids extracted from the reaction mixtures were separated by TLC. Purified BSA was incubated under the same conditions as a control.

Figure 5. PIPKIγ_i4 is a nuclear-targeted phosphoinositide kinase

(A) MCF10A or (B) HeLa cells were grown on coverslips, fixed in methanol, and probed with antibodies for pan-PIPKIγ (green), PIPKIγ_i4 (Iγ_i4; green), E-cadherin (ECD) (red), and SC-35 (red). Co-localization of red and green immunofluorescence channels is indicated in yellow. DAPI (4′,6-diamidino-2-phenylindole) staining was omitted from merged images (Merge) of labelled nuclei. The scale bar represents 10 μm. (C) The cytosolic (Cyto) and nuclear (Nuc) protein fractions of HeLa cells were separated as described in the Experimental section, then subject to Western blotting with anti-PIPKIγ_i4 and pan-PIPKIγ antibodies. Lamin β1 and β-tubulin were Western-blotted as controls for the nuclear and cytosolic fractions respectively. Abbreviation: WCL, whole-cell lysate.

Figure 6. PIPKIγ_i5 localizes to endosomal compartments

HeLa cells expressing HA-tagged PIPKIγ_i5 were fixed in paraformaldehyde and stained with anti-HA (green) and antibodies towards TfnR (recycling endosomes), EEA1 (early endosomes), CD63 (multi-vesicular bodies/late endosomes) and LAMP-1 (lysosomes) (all in red). Yellow areas indicate co-localization of red and green immunofluorescence signals. The inset figures are 175% zooms of the outlined area. The scale bar represents 10 μm.

Figure 7. Localization of PIPKIγ_i5 is distinct from that of PIPKIγ_i2

HeLa cells expressing HA-tagged PIPKIγ_i5 were fixed in paraformaldehyde and stained with (A) anti-HA (green) and anti-talin (red) or (B) anti-HA (green) and anti-N-cadherin (NCD) (red) antibodies. Yellow areas indicate co-localization of red and green immunofluorescence signals. The inset figures are 175% zooms of the outlined area. The scale bar represents 10 μm.