Phosphorylation at conserved carboxyl-terminal hydrophobic motif regulates the catalytic and regulatory domains of protein kinase C.

Alexandra C. Newton,Amelia S. Edwards

doi:10.1074/jbc.272.29.18382

Alexandra C. Newton, Amelia S. Edwards

Open Access

https://doi.org/10.1074/jbc.272.29.18382

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Abstract

Mature protein kinase C is phosphorylated at a conserved carboxyl-terminal motif that contains a Ser (or Thr) bracketed by two hydrophobic residues; in protein kinase C betaII, this residue is Ser-660 (Keranen, L. M., Dutil, E. M., and Newton, A. C. (1995) Curr. Biol. 5, 1394-1403). This contribution examines how negative charge at this position regulates the function of protein kinase C. Specifically, Ser-660 in protein kinase C betaII was mutated to Ala or Glu and the enzyme's stability, membrane interaction, Ca2+ regulation, and kinetic parameters were compared with those of wild-type protein phosphorylated at residue 660. Negative charge at this position had no significant effect on the enzyme's diacylglycerol-stimulated membrane interaction nor the conformational change accompanying membrane binding. In contrast, phosphate caused a 10-fold increase in the enzyme's affinity for Ca2+ and a comparable increase in its affinity for phosphatidylserine, two interactions that are mediated by the C2 domain. Negative charge also increased the protein's thermal stability and decreased its Km for ATP and peptide substrate. These data indicate that phosphorylation at the extreme carboxyl terminus of protein kinase C structures the active site so that it binds ATP and substrate with higher affinity and structures determinants in the regulatory region enabling higher affinity binding of Ca2+. The motif surrounding Ser-660 in protein kinase C betaII is found in a number of other kinases, suggesting interactions promoted by phosphorylation of the carboxyl terminus may provide a general mechanism for stabilizing kinase structure.

Highlights

Phosphorylation is a widely used mechanism for reversibly regulating protein structure and function
Best characterized is the requirement for negative charge on the activation loop of kinases that renders the kinase core competent for catalysis [3, 4]
Based on distinct electrophoretic mobility shifts resulting from these phosphorylations and the use of antibodies selective for dephosphorylated Ser660, the order of phosphorylations was established to be Thr500, followed by Thr-641, followed by Ser-660 [5]. Each of these phosphorylatable residues has an analogue in other protein kinases: Thr-500 aligns with the activation loop phosphorylatable residue found in many protein kinases (including Thr-197 in protein kinase A [4]), Thr-641 has an analogous site on protein kinase A (S338) [9], and Ser-660 is part of a hydrophobic phosphorylation motif present in diverse kinases such as S6 kinase and protein kinase B (Akt kinase) that has the consensus sequence FXXF(S/T)(F/Y) [10, 11]

Summary

Introduction

Phosphorylation is a widely used mechanism for reversibly regulating protein structure and function. Phosphorylation has recently been shown to play an essential role in regulating the protein kinase Cs [5, 6] These enzymes transduce the myriad of signals promoting phospholipid hydrolysis [7]. Based on distinct electrophoretic mobility shifts resulting from these phosphorylations and the use of antibodies selective for dephosphorylated Ser660, the order of phosphorylations was established to be Thr500, followed by Thr-641, followed by Ser-660 [5] Each of these phosphorylatable residues has an analogue in other protein kinases: Thr-500 aligns with the activation loop phosphorylatable residue found in many protein kinases (including Thr-197 in protein kinase A [4]), Thr-641 has an analogous site on protein kinase A (S338) [9], and Ser-660 is part of a hydrophobic phosphorylation motif present in diverse kinases such as S6 kinase and protein kinase B (Akt kinase) that has the consensus sequence FXXF(S/T)(F/Y) [10, 11]. This paper is available on line at http://www.jbc.org loop sequence, does not require phosphorylation of the residue corresponding to Thr-500 in protein kinase C ␤II for the formation of mature, activable enzyme [20]

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