Michael J. Schell

Hepatocyte plasma membrane ecto-ATPase ( pp120 HA4) is a substrate for tyrosine kinase activity of the insulin receptor

pp120/HA4, a membrane protein found in hepatocyte plasma membranes and a substrate for the insulin receptor tyrosine kinase, was purified to homogeneity, subjected to partial proteolysis, and peptides were sequenced by Edman degradation. Six amino acid sequences were obtained, and they matched the deduced amino acid sequences of six regions of a hepatocyte membrane protein called ecto-ATPase.

The Rapamycin and FKBP12 Target (RAFT) Displays Phosphatidylinositol 4-Kinase Activity

The immunosuppressant rapamycin prevents cell cycle progression in several mammalian cell lines and the yeast Saccharomyces cerevisiae. In mammalian cells, rapamycin binds to the small FK506-binding protein, FKBP12, allowing the drug-receptor complex to interact with the 289-kDa RAFT1/FRAP proteins. These proteins, along with their yeast homologs, TOR1/DRR1 and TOR2/DRR2, contain a C-terminal domain with amino acid homology to several phosphatidylinositol (PI) 4- and 3-kinases. However, no direct demonstration of kinase activity for this family of proteins has been reported. We now show that RAFT1, immunoprecipitated from rat brain and MG63 and HEK293 cells, contains PI 4-kinase activity and that rapamycin-FKBP12 has no effect on this activity. Thus, it is likely that, in vivo, rapamycin does not directly inhibit the PI 4-kinase activity and affects the RAFT1/FRAP protein through another mechanism.

Regulation of Inositol 1,4,5-Trisphosphate 3-Kinases by Calcium and Localization in Cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinases (IP3Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P3 into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca2+ signaling, and how, is still not fully understood. In this study, we have used a simple Ca2+ imaging assay to compare the abilities of various Ins (1,4,5)P3-metabolizing enzymes to regulate a maximal histamine-stimulated Ca2+ signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP3K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca2+ release stimulated by 100 μm histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP3Ks to dampen the Ins (1,4,5)P3-mediated Ca2+ signals. We also compared the effects of the these IP3Ks with other enzymes that metabolize Ins(1,4,5)P3, including the Type I Ins(1,4,5)P3 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP3K found in Drosophila. All reduce the Ca2+ signal but to varying degrees. We demonstrate that the activity of only one of two IP3K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP3Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P3 signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.