Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence

Abstract

The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCalpha-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

Scanning Mutagenesis Studies Reveal Multiple Distinct Regions within the Human Protein Kinase C Alpha Regulatory Domain Important for Phorbol Ester-dependent Activation of the Enzyme

“…37 By assessing the ability of these alanine-scanning mutant regulatory domains to inhibit PKC catalytic activity, we were able to identify regions within the PKC regulatory domain lying outside of the pseudosubstrate sequence that are important for inhibition of catalytic domain activity consistent with other studies. 33,38 By fusing each of these constructs inframe with the entire PKC catalytic domain, we have been able to assess, in this study, the role of various regions within PKC for phorbol ester binding and for phorbol-dependent activation of the enzyme.…”

Conserved Modular Domains Team up to Latch-open Active Protein Kinase Cα

“…1a) tethered with (Gly-Ser-Gly) 2-4 linkers to allow for rotational freedom. Basally within a cell or in the presence of EGTA in vitro, the RDs (includes N-to-C-terminal pseudosubstrate, C1a, C1b, and C2 domains) of PKC are known to engage in cis inhibitory intramolecular interactions with the CD (includes the kinase domain and the C-terminal extension termed the C-tail) (18,22). Furthermore, the small molecules Ca 2ϩ , DAG/ PMA, and BimI have been established to interact primarily with the C2, C1, and catalytic domains, respectively (6).…”

The link between PKCα regulation and cellular transformation

“…The 'primed' state of PKC␣ is maintained, at least in part, by the presence of an N-terminal autoinhibitory, pseudosubstrate sequence that occupies the active site within the catalytic domain rendering it unable to bind and phosphorylate substrate [1]. However, it is clear that other regions of the regulatory domain contribute towards the autoinhibition of PKC␣ activity, as removal of the pseudosubstrate domain only partially relieves the inhibition of PKC activity [12].…”