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EXPRESSION AND ACTIVATION OF CARDIAC PROTEIN KINASES N1 AND N2 (PKN1/2)
  1. S Osborne,
  2. SJ Fuller,
  3. PH Sugden,
  4. A Clerk
  1. School of Biological Sciences, University of Reading

Abstract

Rationale PKN1 is associated with cardiac hypertrophy and cardioprotection. The expression/role of the related kinase, PKN2, in the heart is unclear. We compared the expression of PKN1 and 2 in cardiomyocytes and hearts to determine their likely significance, and examined whether they are similarly activated.

Methodology Expression of PKN1/PKN2 mRNA and protein was assessed during postnatal development of rat hearts and in neonatal/adult ventricular myocytes using microarrays and immunoblotting. Effects of agonist stimulation of cardiomyocytes on phosphorylation was assessed by band shifts on immunoblots.

Results PKN1/PKN2 mRNAs were detected in 2d and adult cardiomyocytes. PKN1 protein expression was 2.8-fold and 3.4-fold higher than PKN2 in 2d and adult hearts, respectively, and both declined in adult relative to 2d hearts (2d: 0.33±0.02 vs 0.12±0.04 fmol/µg protein; adult: 0.25±0.02 vs 0.08±0.01 fmol/µg protein). In neonatal or adult cardiomyocytes, PKN2 expression was substantially higher than PKN1 (112±7 vs 56±2 fmol/10^6 cells at 2d; 1188±367 vs 492±151 fmol/10^6 cells in adults) suggesting that PKN2 is enriched in cardiomyocytes. Exposure of cardiomyocytes to the PP1/PP2A inhibitor, calyculin A, produced reduced mobility bands for PKN1 and 2 on immunoblots, indicative of phosphorylation. Epidermal growth factor induced a small mobility shift for PKN1/PKN2 at 5–10 min which returned to baseline by 30 min.

Conclusion These data indicate that, whilst PKN1 plays an important role in the heart, expression levels of PKN2 are substantially higher in cardiomyocytes and both are phosphorylated in response to EGF. Thus, PKN2 is likely to play a significant role in cardiac pathologies.

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