CLONING OF A HUMAN INSULIN-STIMULATED PROTEIN-KINASE (ISPK-1) GENE AND ANALYSIS OF CODING REGIONS AND MESSENGER-RNA LEVELS OF THE ISPK-1 AND THE PROTEIN PHOSPHATASE-1 GENES IN MUSCLE FROM NIDDM PATIENTS

Complementary DNA encoding three catalytic subunits of protein phospha tase 1 (PP1 alpha, PP1 beta, and PP1 gamma) and the insulin-stimulated protein kinase 1 (ISPK-1) was analyzed for variations in the coding r egions related to insulin-resistant glycogen synthesis in skeletal mus cle of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM ). The human ISPK-1 cDNA was cloned hom T-cell leukemia and placental cDNA libraries and mapped to the short arm of the human X chromosome. Single-strand conformation polymorphism (SSCP) analysis identified a t otal of six variations in the coding regions of the PP1 genes: two in PP1 alpha. at codons 90 and 255; one in PP1 beta at codon 67; and thre e in PP1 gamma at codons 11, 269, and 273, respectively. All mere, how ever, silent single nucleotide substitutions. SSCP analysis of the ISP K-1 gene identified one silent polymorphism at codon 266 and one amino acid variant at codon 38 (Ile-->Ser). This variant was primarily foun d in one male NIDDM patient. This subject, however, did not exhibit an impairment of muscle insulin-stimulated glycogen synthase activation. No significant differences were found in mRNA levels in muscle of the four genes between 15 NIDDM patients and 14 healthy subjects. Our fin dings suggest that 1) genetic abnormalities in the coding regions of P P1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are unlikely to be frequentl y occurring causes of the reduced insulin-stimulated activation of the glycogen synthesis in muscle from the analyzed group of NIDDM patient s; 2) the mRNA levels of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 ar e normal in muscle horn the NIDDM patients; and 3) putative inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstre am of ISPK-1 in the insulin action cascade.