Autoregulation of Enzymes by Pseudosubstrate Prototopes: Myosin Light Chain Kinase

Pearson, Richard B.; Wettenhall, Richard E.H.; Means, Anthony R.; Hartshorne, David J.; Kemp, Bruce E.

doi:10.1126/science.3406746

Cited by 151 publications

(75 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Activation in response to signal-transduction factors arises by the relief of inhibition via the dissociation of the pseudosubstrate domain and the consequential availability of the catalytic domain to then productively bind the protein substrate. This mechanism was first elucidated for the cAMP-dependent protein kinase (PKA) [33] and subsequently shown to be the mechanism of regulation of many protein kinases [34,35]. This mechanism is abundantly evident in PhK, where the catalytic activity of the Phk γ subunit is markedly reduced by the binding of the α, β and δ subunits when present as the (αβγδ) % holoenzyme, or in the partial subcomplexes αγδ and γδ.…”

Section: Discussionmentioning

confidence: 99%

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

O’Mahony

Walsh

2002

Biochem. J.

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

O’Mahony

Walsh

2002

Biochem. J.

View full text Add to dashboard Cite

“…The PKC activation induced by PtdSer/phorbol ester binding could be therefore the consequence of conformational changes resulting in the release of the pseudosubstrate region from the catalytic site. Studies concerning the mechanism of activation of other allosterically regulated kinases, for example myosin light chain kinase [33], show that the pseudosubstrate site plays an important role in the autoregulation of the enzyme. PKCy, in its activated state, conserves the ability to interact with the 15G4 mAb, indicating that the 15G4 epitope (297-310 sequence within the hinge region V3) remains accessible after the conformational changes induced by the binding of cofactors.…”

Section: S O G T W E E~s K F D~~~~~g S F G~s E R K G~e L Y A~~~q D D D~cmentioning

confidence: 99%

Effector‐dependent conformational changes in protein kinase Cγ through epitope mapping with inhibitory monoclonal antibodies

Cazaubon

Webster

Camoin

et al. 1990

European Journal of Biochemistry

View full text Add to dashboard Cite

Three monoclonal antibodies (mAb) directed against the regulatory domain of the protein kinase Cy (PKCy); 15G4, 5A2 and 3669, were shown to display distinct properties with respect to PKCy kinase activity [Cazaubon, S., Marais, R., Parker, P. & Strosberg, A.D. (1989) Eur. J . Biochem. 182, 401 -4061. The mAb 5A2 and 3669, which act as potent inhibitors of the cofactor-dependent kinase activity, can no longer bind PKCy in the presence of phosphatidylserine and phosphatidylserine/phorbol ester, respectively; 1564 binding is not influenced by effectors. Due to this functional relationship between the inhibitory mAb-and cofactor-binding sites, we sought to localize the mAb epitopes with respect to the functional sites of PKCy. For this purpose, several deletions were introduced at the 5' end of the PKCy cDNA and the mutant proteins were expressed in Escherichia coli. The determination of the immunoreactivity of the deleted PKCy proteins shows that the amino acid residues essential to the binding of 5A2 and 3669 are directly adjacent to the second cysteine-rich motif: these are contained in the sequences at positions 151 -163 and 164-197, respectively. In addition, various deletions around the C1 region of the regulatory domain allowed the identification of the second cysteine-rich motif as a functional binding site for phorbol dibutyrate. These deletion studies thus demonstrate that the epitopes recognized by the inhibitory mAbs 5A2 and 3669 are distinct from the cofactor-binding sites. This suggests that the binding of phosphatidylserine and phorbol ester induce conformational changes in the regulatory domain of PKC, which are thus responsible for the loss of the 5A2 and 3669 immunoreactivity of the native protein. In this conformational state, PKCy conserves its ability to interact with the non-inhibitory mAb 15G4. By using synthetic peptides, the 1564 epitope was localized to the sequence 297 -3 10 in the V3 variable region. This indicates that the flexibility of the V3 region, which delimits the C-terminus of the regulatory domain, may not be necessary for the allosteric activation of PKC.In view of these results, we propose that PKC activation by its cofactors results in intramolecular changes which allow the enzyme to bind exogenous substrates.Protein kinase C (PKC) has attracted much attention because of its important role in the transduction of various external signals including hormones, growth factors, neuropeptides and antigens [l]. Originally defined as a Ca2+-and phospholipid-dependent protein kinase [2], PKC is physiologically regulated by the second messenger diacylglycerol [3, 41. Stimulation of receptors coupled to phospholipase C, induces PKC activation through diacylglycerol formation from ionositol phospholipids. In addition, PKC has been identified as the major intracellular receptor for biologically active tumor promotors of the phorbol ester class such as 12-0-tetradecanoylphorbol13-acetate (TPA) and phorbol12,13-dibutyrate, which function as analogs of diacylglycerol [5, 61. While signific...

show abstract

“…For the synthetic peptide studies the kinase was assayed in 40 p1 40 mM Hepes, pH 7.0, containing 5 mM magnesium acetate, 0.25 mM [Y-~~PIATP (500 cpm/pmol), 0.1 mM EGTA, 0.55 mM CaClZ, calmodulin and synthetic peptide substrate [Argl2, Ala14, AlalSlMLC (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) at the concentrations indicated. The peptide substrate had the sequence KRRAARATSNVFA.…”

Section: Mlck Assaymentioning

confidence: 99%

Proteolytic cleavage sites in smooth muscle myosin‐light‐chain kinase and their relation to structural and regulatory domains

Pearson¹,

M²,

Morrice³

et al. 1991

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

Proteolysis of the smooth muscle myosin-light-chain kinase with either thermolysin or endoproteinase Lys-C cleaves the enzyme towards the amino-terminus between the first and second unc domains, unc-11-1 and unc-11-2, and in the calmodulin-binding domain. The thermolytic fragment extends 532 residues from Ser275 to Ala806 and is resistant to further digestion. It is catalytically inactive and does not bind calmodulin. Further proteolysis of the thermolytic fragment with trypsin generates a constitutively active fragment. Digestion with endoproteinase Lys-C initially results in an inactive fragment of 516 residues, Ala287 to Lys802. Further digestion with Lys-C endoproteinase results in a constitutively active 474-residue fragment with the same amino-terminus, but a carboxyl-terminus at Lys760, near Arg762, the last conserved residue of protein kinase catalytic domains. There is no cleavage in the acidic-residue-rich connecting peptide between the amino-terminus of the catalytic domain and the unc-I domain, nor within the unc-I1 or unc-I domains or between the adjacent unc-11-2 and unc-I domains. The pattern of cleavages by these proteases reflects well the predicted domain structure of the myosin-light-chain kinase and further delineates the regulatory pseudosubstrate region. A synthetic peptide corresponding to the pseudosubstrate sequence, MLCK(787 -807) was a more potent inhibitor by three orders of magnitude than the overlapping peptide MLCK(777-793) proposed by Ikebe et al.

show abstract

Autoregulation of Enzymes by Pseudosubstrate Prototopes: Myosin Light Chain Kinase

Cited by 151 publications

References 22 publications

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

Differentiation-dependent mechanisms of transcriptional regulation of the catalytic subunit of phosphorylase kinase

Effector‐dependent conformational changes in protein kinase Cγ through epitope mapping with inhibitory monoclonal antibodies

Proteolytic cleavage sites in smooth muscle myosin‐light‐chain kinase and their relation to structural and regulatory domains

Contact Info

Product

Resources

About