Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or ~) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an Mr 66 138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, an SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

In a panel of large Caucasian pedigrees, we genotyped markers in eight chromosomal regions previously reported as supporting linkage with type 2 diabetes. We previously reported significant linkage on chromosome 20q (maximum logarithm of odds score [MLS] ‫؍‬ 2.79) in this panel. In the present analysis, candidate regions on 1q, 2q, 3q, 5q, 9q, and 10q yielded little evidence for linkage; a region on 2p (MLS ‫؍‬ 1.64, P ‫؍‬ 0.01 at position 9.0 cM) gave suggestive evidence of linkage; and a region on 8p (MLS ‫؍‬ 3.67, P ‫؍‬ 2.8 ؋ 10 ؊5 , at position 7.6 cM) gave significant evidence of linkage. Conditional analyses were performed for both 2p and 8p regions and the region reported on 20q. The MLS for 2p increased from 1.64 to 1.79 (empirical P ‫؍‬ 0.142) when conditioned for heterogeneity on 20q. The case was similar for 8p, where the MLS increased from 3.67 to 4.51 (empirical P ‫؍‬ 0.023) when conditioned on families without evidence of linkage at 20q. In conclusion, our data support a type 2 diabetes susceptibility locus on chromosome 8p that appears to be independent from other susceptibility loci. Although we were able to replicate linkage in our pedigrees on chromosome 2p, we did not find evidence of linkage for regions on 1q, 2q, 3q, 5q, 9q, or 10q. Diabetes 53: 486 -491, 2004 T ype 2 diabetes is a disease characterized by hyperglycemia, insulin resistance, and impaired insulin secretion (1). Although the pathophysiology of this disease is not well understood, evidence of familial clustering indicates that genetic susceptibility plays a role in disease development (2,3). Identification of genetic susceptibility factors, however, has been difficult, and, to date, the putative genes contributing to the etiology of type 2 diabetes remain unknown.Efforts to identify the genetic factors underlying susceptibility to type 2 diabetes have used both candidate gene approaches and linkage studies (4). More than a dozen genomewide scans have been completed from which various chromosomal regions with suggestive or significant evidence of linkage with type 2 diabetes have emerged (5-23). However, despite intense efforts to clone the susceptibility genes, the identification of these genes has been unsuccessful, with the single exception of calpain-10 (CAPN10) or NIDDM1 (24).Efforts to clone type 2 diabetes susceptibility loci have been plagued by inconsistent findings. Evidence for linkage with type 2 diabetes has been reported on most autosomes (25). However, there has been little overlap among linkage results, with the notable exceptions being the findings on chromosomes 1q (8,11,15,19,23) and 20q (26 -30). Factors contributing to the failure to replicate linkage results include the presence of environmentally determined phenocopies of type 2 diabetes as well as the complex nature of the inheritance of type 2 diabetes. The latter includes genetic and allelic heterogeneity, epistasis (gene-to-gene interaction), and gene-to-environment interactions.In a recent study, we showed that the overall sibling genetic risk rati...

A 74-kDa delta/B" subunit was isolated by heparin-Sepharose column chromatography from human erythrocyte protein phosphatase 2A (PP2A) consisting of a 34-kDa catalytic subunit (alpha/C) and 63- and 74-kDa regulatory subunits (beta/A and delta/B") in a ratio of 1:1:1. The purified delta/B" was used as an immunogen in mice, to prepare specific antisera against delta/B". Immunoblot analyses with the antisera detected an immunoreactive 72-kDa protein in the cytosol from various rat tissues including erythrocytes, brain, lung, testis, adrenal gland, heart, spleen, kidney, and liver. The 72-kDa protein was highly abundant in brain and was distributed evenly in cerebral cortex, cerebellum, and brain stem. The 72-kDa protein was also detected in mitochondria and microsome fractions. An immunoreactive 68-kDa protein was detected mainly in nuclear and microsome fractions. The 72-kDa protein from rat brain cytosol copurified with phosphorylated H2B histone phosphatase activity during successive chromatographies on DEAE-Toyopearl, AH-Sepharose, Sephadex G-150, H1 histone-Toyopearl, TSK DEAE-5PW, protamine-Toyopearl, and TSK G3000SW columns. The purified enzyme migrated as a single protein band on nondenaturing PAGE and as three protein bands of 34, 63, and 72 kDa in a ratio of 1:1:1 on SDS-PAGE. The molecular weight of the enzyme was estimated to be 170,000 from the s20,W value of 7.2 +/- 0.3 S and the Stokes radius of 5.5 +/- 0.1 nm. The rat brain enzyme was classified as PP2A, based on the following properties; (1) an IC50 for okadaic acid of 10(-9) M; (2) its preferential dephosphorylation of the a subunit of phosphorylase kinase; (3) its insensitivity to protein inhibitor 2; and (4) its heterotrimeric subunit structure. The Km value and the molecular activity of the enzyme for phosphorylated H2B histone were 72.3 +/- 0.3 microM and 192 +/- 2 mol Pi released/min/mol enzyme, respectively, and were comparable to those of human erythrocyte PP2A (alpha1 beta1 delta1/ CAB"). The 72-kDa subunit in the purified rat brain PP2A was phosphorylated in vitro by cAMP-dependent protein kinase.

A MnP+ -dependent protein phosphatase 2A which is composed of a 34 kDa catalytic CP subunit and a 63 kDa regulatory AP subunit, was purified from human erythrocyte cytosol. CP and AP produced V8-and papain-peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn P+ -independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of CPA and CAP revealed that the metal dependency resided in CP and not in AP. In CA, 0.87 þ 0.12 mol zinc and 0.35 þ 0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in CPAP. Pre-incubation of CP with ZnCl P and FeCl P , but not FeCl Q , synergistically stimulated the Mn P+ -independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn P+ -and Fe P+ -metalloenzyme and that CP is the apoenzyme.z 1999 Federation of European Biochemical Societies.