Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

O’Mahony, Alison; Walsh, Donal A.

doi:10.1042/0264-6021:3620199

Cited by 2 publications

(1 citation statement)

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“…For the three expressed proteins, (αβγδ) 4 , αγδ, and γδ, there is an increase in both the pH 6.8 and the pH 8.2 activity as the proteins get less complex. This is consistent with the γ subunit being less inhibited in the absence of the other subunits, an important feature of the protein that has been described in more detail elsewhere ( , ). Of note, the pH 8.2 activity of the expressed γδ approached close to that of the holoenzyme Phk enzyme purified from skeletal muscle.…”

Section: Resultssupporting

confidence: 90%

Baculovirus-Mediated Overexpression of the Phosphorylase b Kinase Holoenzyme and αγδ and γδ Subcomplexes

et al. 2004

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Recombinant baculoviruses were created and used to coexpress rat phosphorylase kinase (Phk) alpha, gamma, and delta subunits and rabbit beta subunit in insect cells. Coexpression allowed creation of the (alphabetagammadelta)4 hexadecamer, the alphagammadelta heterotrimer, and the gammadelta heterodimeric subcomplexes. Neither the individual alpha, beta, or gamma subunit nor any complex containing the beta subunit other than the hexadecameric holoenzyme was obtained in soluble form. The expressed complexes exhibited pH- and [Ca2+]-dependent specific activities that were similar to those of the Phk holoenzyme purified from rabbit skeletal muscle (SkM Phk). SkM Phk, expressed Phk, and the alphagammadelta subcomplex were activated by exogenous calmodulin and underwent Ca(2+)-dependent autophosphorylation. In some of these features there were subtle differences that could likely be attributed to differences in the covalent modification state of the baculovirus-driven expressed protein. Our results provide an important avenue to probe the detailed characterization of the structure of Phk and the function of the individual domains of the subunits using baculovirus-mediated expression of Phk and Phk subcomplexes.

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Section: Resultssupporting

confidence: 90%