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A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase.

Publication ,  Journal Article
Sale, EM; Hodgkinson, CP; Jones, NP; Sale, GJ
Published in: Biochemistry
January 10, 2006

Protein kinase B appears to play a key role in insulin signaling and in the control of apoptosis, although the precise targets of PKB are incompletely understood. PKB exists as three isoforms (alpha, beta, and gamma) that may have unique as well as common functions within the cell. To facilitate understanding the precise roles of PKB and its isoforms, novel tools of widespread applicability are described. These tools are antisense oligonucleotide probes that enable the specific and potent knock down of endogenous PKB alpha, beta, or gamma isoforms, individually or in various combinations, including concurrent removal of all three isoforms. The probes were applied to dissect the role of PKB in phosphorylating glycogen synthase kinase-3 (GSK-3), a critical mediator in multiple responses, and other potentially key targets. Triple antisense knock down of PKB alpha, beta, and gamma so that total PKB was <6% blocked insulin-stimulated phosphorylation of endogenous GSK-3alpha and GSK-3beta isoforms by 67% and 45%, respectively, showing that GSK-3alpha and GSK-3beta are controlled by endogenous PKB. Each PKB isoform contributed to GSK-3alpha and GSK-3beta phosphorylation, with PKBbeta having the predominant role. Knock down of total PKB incompletely blocked insulin-stimulated phosphorylation of GSK-3alpha and GSK-3beta, and a pathway involving atypical PKCs, zeta/lambda, was shown to contribute to the signal. Triple antisense knock down of PKB alpha, beta, and gamma abrogated the insulin-stimulated phosphorylation of WNK1, ATP citrate lyase, and tuberin. However, antisense-mediated knock down of PKB alpha, beta, and gamma had no effect on insulin-stimulated DNA synthesis in 3T3-L1 adipocytes, indicating that pathways other than PKB mediate this response in these cells. Finally, our PKB antisense strategy provides a method of general usefulness for further dissecting the precise targets and roles of PKB and its isoforms.

Duke Scholars

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

January 10, 2006

Volume

45

Issue

1

Start / End Page

213 / 223

Location

United States

Related Subject Headings

  • WNK Lysine-Deficient Protein Kinase 1
  • Tumor Suppressor Proteins
  • Tuberous Sclerosis Complex 2 Protein
  • Proto-Oncogene Proteins c-akt
  • Protein Serine-Threonine Kinases
  • Protein Kinase C
  • Protein Isoforms
  • Phosphorylation
  • Oligonucleotide Probes
  • Minor Histocompatibility Antigens
 

Citation

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Sale, E. M., Hodgkinson, C. P., Jones, N. P., & Sale, G. J. (2006). A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase. Biochemistry, 45(1), 213–223. https://doi.org/10.1021/bi050287i
Sale, Elizabeth M., Conrad P. Hodgkinson, Neil P. Jones, and Graham J. Sale. “A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase.Biochemistry 45, no. 1 (January 10, 2006): 213–23. https://doi.org/10.1021/bi050287i.
Sale, Elizabeth M., et al. “A new strategy for studying protein kinase B and its three isoforms. Role of protein kinase B in phosphorylating glycogen synthase kinase-3, tuberin, WNK1, and ATP citrate lyase.Biochemistry, vol. 45, no. 1, Jan. 2006, pp. 213–23. Pubmed, doi:10.1021/bi050287i.
Journal cover image

Published In

Biochemistry

DOI

ISSN

0006-2960

Publication Date

January 10, 2006

Volume

45

Issue

1

Start / End Page

213 / 223

Location

United States

Related Subject Headings

  • WNK Lysine-Deficient Protein Kinase 1
  • Tumor Suppressor Proteins
  • Tuberous Sclerosis Complex 2 Protein
  • Proto-Oncogene Proteins c-akt
  • Protein Serine-Threonine Kinases
  • Protein Kinase C
  • Protein Isoforms
  • Phosphorylation
  • Oligonucleotide Probes
  • Minor Histocompatibility Antigens