The broad specificity of dominant inhibitory protein kinase C mutants infers a common step in phosphorylation

Abstract

Dominant negative properties are conferred on protein kinase (PK) Calpha by mutation of the phosphorylation site in the activation loop of the kinase domain. To address the universality and/or specificity of such mutations, analogous alterations were introduced in other members of the PKC family and tested for their effects on the function of co-transfected activated PKC. For all three subclasses of the PKC family, mutations of the predicted activation loop phosphorylation sites resulted in dominant negative properties. These properties were not restricted to the cognate PKC isotypes, but were effective across the different subclasses. For example, two PKCzeta mutants (atypical isotype) inhibited both PKCalpha (classical isotype) and PKCepsilon (novel isotype). For all these mutants, inhibition correlated with an ability to prevent the accumulation of phosphorylated PKCalpha, consistent with the expected mode of action. In the case of the PKCalpha mutant, it was shown that inhibition required the full-length mutant protein. The results provide evidence for the involvement of a common step in the phosphorylation of all PKC isotypes.

Signalling Pathways Regulating the Dephosphorylation of Ser729 in the Hydrophobic Domain of Protein Kinase Cε upon Cell Passage

“…4B). PI 3-kinase has already been demonstrated to play a role in PKC phosphorylation through its activation of phospholipid-dependent kinase 1 (7,8). Our findings also implicate PI 3-kinase in the control of PKC⑀ dephosphorylation and degradation.…”

p70 S6 Kinase Is Regulated by Protein Kinase Cζ and Participates in a Phosphoinositide 3-Kinase-Regulated Signalling Complex

“…Taken together, these data indicate that PKC positively regulates p70S6K through a concerted effect with other p70S6K regulators and that the inhibitory effects of PKC K/W are due to a lack of positive function rather than to sequestration of upstream p70S6K activators. Recently, it was reported that a kinase-inactive mutant of PKC containing a mutation in its activation loop (threonine 410 mutated to alanine) can antagonize PKCε and PKC␣ activities due to a common activation mechanism (20). We found that PKC K/W did not affect activation of p70S6K by PMA (data not shown), which activates conventional and/or novel PKCs but not atypical isoforms, such as PKC and -.…”

Cell Volume Regulation in Response to Hypotonicity Is Impaired in HeLa Cells Expressing a Protein Kinase C α Mutant Lacking Kinase Activity

“…8). The control using a corresponding PKC mutant had no consequence on G Cl,swell , thereby eliminating the possibility of a promiscuous effect, for instance, due to sequestration of upstream kinases such as PDK1, essential for activation of several PKCs (52). It should be noted that the PKC mutant displayed slower G Cl,swell activation in comparison to non-transfected cells (see Fig.…”