Expressed phosphorylase b kinase and its alphagammadelta subcomplex as regulatory models for the rabbit skeletal muscle holoenzyme.

Understanding the regulatory interactions among the 16 subunits of the (alphabetagammadelta)(4) phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells alpha, beta, gamma, and delta subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding alphagammadelta trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and alphagammadelta exhibited only a very low Ca(2+)-independent activity and a Ca(2+)-dependent activity similar to that of the native holoenzyme with [Ca(2+)](0.5) of 0.4 microM for the RM PhK, 0.7 microM for the rPhK, and 1.5 microM for the alphagammadelta trimer. The RM PhK, rPhK, and alphagammadelta subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the alpha-gamma intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant alphagammadelta subcomplex. Our results indicate that both the rPhK and alphagammadelta subcomplex are promising models for future structure-function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.

The regulatory beta subunit of phosphorylase kinase interacts with glyceraldehyde-3-phosphate dehydrogenase.

Skeletal muscle phosphorylase kinase (PhK) is an (alphabetagammadelta) 4 hetero-oligomeric enzyme complex that phosphorylates and activates glycogen phosphorylase b (GP b) in a Ca (2+)-dependent reaction that couples muscle contraction with glycogen breakdown. GP b is PhK's only known in vivo substrate; however, given the great size and multiple subunits of the PhK complex, we screened muscle extracts for other potential targets. Extracts of P/J (control) and I/lnJ (PhK deficient) mice were incubated with [gamma- (32)P]ATP with or without Ca (2+) and compared to identify potential substrates. Candidate targets were resolved by two-dimensional polyacrylamide gel electrophoresis, and phosphorylated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified by matrix-assisted laser desorption ionization mass spectroscopy. In vitro studies showed GAPDH to be a Ca (2+)-dependent substrate of PhK, although the rate of phosphorylation is very slow. GAPDH does, however, bind tightly to PhK, inhibiting at low concentrations (IC 50 approximately 0.45 microM) PhK's conversion of GP b. When a short synthetic peptide substrate was substituted for GP b, the inhibition was negligible, suggesting that GAPDH may inhibit predominantly by binding to the PhK complex at a locus distinct from its active site on the gamma subunit. To test this notion, the PhK-GAPDH complex was incubated with a chemical cross-linker, and a dimer between the regulatory beta subunit of PhK and GAPDH was formed. This interaction was confirmed by the fact that a subcomplex of PhK missing the beta subunit, specifically an alphagammadelta subcomplex, was unable to phosphorylate GAPDH, even though it is catalytically active toward GP b. Moreover, GAPDH had no effect on the conversion of GP b by the alphagammadelta subcomplex. The interactions described herein between the beta subunit of PhK and GAPDH provide a possible mechanism for the direct linkage of glycogenolysis and glycolysis in skeletal muscle.

Dynamics of the metal-dependent transcription factor complex in vivo at the mouse metallothionein-I promoter.

The in vivo association of transcription factors with the metallothionein-I promoter was examined using chromatin immunoprecipitation (ChIP) assays. The results demonstrated that c-fos is rapidly recruited along with the metal response element-binding transcription factor-1 (MTF-1) to this promoter in response to zinc or cadmium, and that this recruitment is reversed in the visceral yolk sac by a zinc-deficient diet in vivo, and in cultured cells after lowering the zinc concentration in the medium or during prolonged zinc exposure. In contrast, the interactions of c-jun, USF-1, USF-2 and Sp1 with this promoter are metal-independent. Studies of knockout cells revealed that the recruitment of c-fos to the MT-I promoter requires MTF-1, but that c-fos is not essential for recruitment of MTF-1 and metal-induction of MT-I gene expression. Studies of Hepa cells stably-transfected with reporter genes driven by the MT-I promoter suggested two in vivo binding sites for USF-1 and -2. In contrast, Sp1 was apparently associated with a single binding site (upstream of -153 bp). In addition, maximal recruitment of c-fos by metals required sequences and/or other proteins that interact upstream of -153 bp. In summary, these studies extend our understanding of the complexity and dynamics of the transcription factor complex that forms at the MT-I promoter in vivo in response to metals.

Putative zinc-sensing zinc fingers of metal-response element-binding transcription factor-1 stabilize a metal-dependent chromatin complex on the endogenous metallothionein-I promoter.

The metalloregulatory functions of metal-response element-binding transcription factor-1 (MTF-1) have been mapped, in part, to its six highly conserved zinc fingers. Here we examined the ability of zinc finger deletion mutants of mouse MTF-1 to regulate the endogenous metallothionein-I (MT-I) gene in cells lacking endogenous MTF-1. MTF-1 knockout mouse embryo fibroblasts were transfected with expression vectors for FLAG-tagged MTF-1 (MTF-1flag) or finger deletion mutants of MTF-1flag and then assayed for metal induction of MT-I gene expression, nuclear translocation, and in vitro DNA-binding activity of MTF-1 and its stable association with the endogenous chromosomal MT-I promoter. Intact MTF-1flag restored metal responsiveness of the MT-I gene, underwent nuclear translocation, displayed increased in vitro DNA binding in response to zinc and less so to cadmium, and rapidly formed a stable complex with the MT-I promoter chromatin in response to both of these metals. In contrast, although deletion of finger 1, fingers 5 and 6, or finger 6 only had variable effects on the nuclear localization and in vitro DNA-binding activity of MTF-1, each of these finger-deletion mutants severely attenuated metal-induced MTF-1 binding to the MT-I promoter chromatin and activation of the endogenous MT-I gene. These results demonstrated that the metal-induced recruitment of MTF-1 to the MT-I promoter is a rate-limiting step in its metalloregulatory function and that an intact zinc finger domain is required for this recruitment. During the course of these studies, it was discovered that mouse MTF-1 is polymorphic. The impact of these polymorphisms on MTF-1 metalloregulatory functions is discussed.

Mammalian metal response element-binding transcription factor-1 functions as a zinc sensor in yeast, but not as a sensor of cadmium or oxidative stress.

The zinc finger protein, metal response element-binding transcription factor-1 (MTF-1) regulates the expression of genes in response to metal ions and oxidative stress. The precise mechanisms by which this occurs are not understood. To further examine this problem, mouse MTF-1 was expressed in Saccharomyces cerevisiae and tested for the ability to activate metal response element-driven reporter gene expression. Zinc was an effective inducer of reporter gene expression. In general, the magnitude of zinc induction was dependent on the concentration of zinc in the culture medium, but independent of the amount of MTF-1 expression. Zinc induction also occurred with either integrated or episomal reporter plasmids containing the native mouse metallothionein-I proximal promoter. Deletion of fingers 5 and 6 of MTF-1, which function in a zinc-dependent manner to stabilize the DNA-binding activity of the protein in vitro, did not diminish the zinc induction of either episomal or integrated promoters. However, a Gal4 DNA-binding domain- MTF-1 fusion protein, which binds constitutively to the Gal4-responsive promoter, was not zinc inducible but caused constitutive activation of reporter gene expression. This suggests that zinc activation of the DNA-binding activity of MTF-1 is the rate limiting step in its metalloregulatory function in yeast. In contrast, MTF-1 was not responsive to either cadmium or hydrogen peroxide, suggesting that distinct co-activators or signal transduction cascades not found in yeast are required to mediate MTF-1 activation of gene expression by this toxic metal and by oxidative stress.