Identification of a WD40 repeat-containing isoform of PHIP as a novel regulator of beta-cell growth and survival.
The pleckstrin homology domain-interacting protein (PHIP) was originally identified as a 902-amino-acid (aa) protein that regulates insulin receptor-stimulated GLUT4 translocation in skeletal-muscle cells. Immunoblotting and immunohistological analyses of pancreatic beta-cells reveal prominent expression of a 206-kDa PHIP isoform restricted to the nucleus. Herein, we report the cloning of this larger, 1,821-aa isoform of PHIP (PHIP1), which represents a novel WD40 repeat-containing protein. We demonstrate that PHIP1 overexpression stimulates insulin-like growth factor 1-dependent and -independent proliferation of beta-cells, an event which correlates with transcriptional upregulation of the cyclin D2 promoter and the accumulation of cyclin D2 protein. RNA interference knockdown of PHIP1 in INS-1 cells abrogates insulin receptor substrate 2 (IRS2)-mediated DNA synthesis, providing for a specific role for PHIP1 in the enhancement of IRS2-dependent signaling responses leading to beta-cell growth. Finally, we provide evidence that PHIP1 overexpression blocks free fatty acid-induced apoptosis in INS-1 cells, which is accompanied by marked activation of phosphoprotein kinase B (PKB)/AKT and the concomitant inhibition of caspase-9 and caspase-3 cleavage. Our finding that the restorative effect of PHIP1 on beta-cell lipotoxicity can be attenuated by the overexpression of dominant-negative PKB suggests a key role for PKB in PHIP1-mediated cytoprotection. Taken together, these findings provide strong support for PHIP1 as a novel positive regulator of beta-cell function. We suggest that PHIP1 may be involved in the induction of long-term gene expression programs to promote beta-cell mitogenesis and survival.
Podcheko
Alexey
A
Department of Laboratory Medicine and Pathobiology, 1 King's College Circle, Room 6238, University of Toronto, Toronto, Canada M5S 1A8.
Northcott
Paul
P
Bikopoulos
George
G
Lee
Andrew
A
Bommareddi
Swaroop R
SR
Kushner
Jake A
JA
Farhang-Fallah
Janet
J
Rozakis-Adcock
Maria
M
D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007211:Indoles; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D020033:Protein Isoforms; D034622:RNA Interference; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D005456:Fluorescent Dyes; D007211:Indoles; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526298:Irs2 protein, mouse; D009419:Nerve Tissue Proteins; C420744:Phip protein, mouse; D010750:Phosphoproteins; D020033:Protein Isoforms; C007293:DAPI; D007334:Insulin-Like Growth Factor I; D008156:Luciferases