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J. Biol. Chem., Vol. 279, Issue 12, 11672-11679, March 19, 2004

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Identification and Characterization of a Phosphoinositide Phosphate Kinase Homolog^*

James D. Chang¶, Seth J. Field||, Lucia E. Rameh**, Christopher L. Carpenter, and Lewis C. Cantley

From the Beth Israel Deaconess Medical Center, Divisions of Signal Transduction, Cardiovascular Medicine, and Hematology-Oncology, Boston, Massachusetts 02215, ^||Massachusetts General Hospital, Endocrine Unit, Boston, Massachussetts 02114, ^**Boston Biomedical Research Institute, Watertown, Massachusetts 02472, and Departments of Cell Biology and Medicine, Harvard Medical School, Boston, Massachusetts 02215

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125–1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104–R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types I, I, and I) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) without a significant net increase in total PI(4,5)P₂. When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P₃ production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKI. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P₃.

Received for publication, September 2, 2003 , and in revised form, December 28, 2003.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY376879.

^* This work was supported by National Institutes of Health Grants HL03313 (to J. D. C.), GM54389 (to C. L. C.), and GM36624 (to L. C. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 617-667-0933; Fax: 617-667-0957; E-mail: jchang{at}bidmc.harvard.edu.

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