Tryptic digestion of myosin light chain kinase produces an inactive fragment that is activated on continued digestion

Foster, Carolyn; Fleet, Melinda Van; Marshak, Ann

doi:10.1016/0003-9861(86)90371-1

Cited by 15 publications

(5 citation statements)

References 6 publications

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“…The kinetic parameters for this reaction were quite comparable to those observed for the reaction of FSBA with other protein kinases (Table II). The K{ for MgATP, 34 µ , compared quite favorably with the reported Km values for smMLCK-CaM of 30-70 µ (Hartshorne & Mrwa, 1980;Foster et al, 1986).…”

Section: Discussionsupporting

confidence: 72%

“…As can be seen in Table I, MgATP blocked inactivation of both smMLCK and smMLCK-CaM by FSBA. MgGTP, a purine nucleotide triphosphate that does not function as a phosphoryl donor substrate for smMLCK, offered little protection against catalytic inactivation even at a concentration that was at least 100 times higher than the enzyme's Km for MgATP (Hartshorne & Mrwa, 1980;Foster et al, 1986). The addition of saturating concentrations of a substrate peptide, KKRAAR-ATSNVF (Kemp & Pearson, 1985), had no effect on the reaction.…”

Section: Resultsmentioning

confidence: 99%

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The magnesium-ATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme

Kennelly¹,

Leng²,

Marchand³

1992

Biochemistry

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An ATP-like affinity labeling reagent, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), was used to probe the MgATP-binding site of smooth muscle myosin light chain kinase from chicken gizzard (smMLCK) and its calmodulin (CaM) complex. Native smMLCK has an absolute requirement for the binding of the calcium complex of CaM for expression of its catalytic activity. FSBA reacted with smMLCK-CaM and with the CaM-free, inactive enzyme as well. Both reactions were dependent on time and FSBA concentration. Reaction was accompanied by the incorporation of covalently bound [14C]FSBA into smMLCK protein at a molar ratio of approximately 1:1 in each case. p-(Fluorosulfonyl)benzoic acid, an analogue of FSBA lacking the adenosine targeting group, did not react at a significant rate with either form of smMLCK. Reaction of CaM-free and CaM-bound smMLCK with FSBA displayed saturation kinetics. The first-order rate constants for the conversion of the reversible, noncovalent enzyme-FSBA complex to form the irreversibly inhibited, covalently modified enzyme were similar for both smMLCK and smMLCK-CaM, 0.15 and 0.07 min-1, respectively. The concentrations of FSBA yielding the half-maximal rate of inactivation, KI, were essentially identical--0.65 and 0.64 mM, respectively--for smMLCK and smMLCK-CaM. MgATP, but not MgGTP or a substrate peptide, potently inhibited reaction with FSBA. Inhibition by MgATP was competitive. The measured inhibitory constant for MgATP was essentially the same--33 versus 34 microM--for both smMLCK and smMLCK-CaM. It therefore is concluded that the MgATP-binding site on smMLCK remains accessible and recognizable as such when the enzyme becomes inactivated upon dissociation of CaM.

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Section: Discussionsupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

The magnesium-ATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme

Kennelly¹,

Leng²,

Marchand³

1992

Biochemistry

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“…Limited tryptic digestion of the MLCK (130 kD) for approximately 1 min generates a 64-kD fragment that has lost the capacity to bind calmodulin and has little or no catalytic activity (6,9). Further digestion of 19 AUGUST I988 the enzyme yields a 61-kD fragment that is constitutively active.…”

Section: Iinases Are Involved Inmentioning

confidence: 99%

Autoregulation of Enzymes by Pseudosubstrate Prototopes: Myosin Light Chain Kinase

Pearson¹,

Wettenhall²,

Means³

et al. 1988

Science

151

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The myosin light chain kinase requires calmodulin for activation. Tryptic cleavage of the enzyme generates an inactive 64-kilodalton (kD) fragment that can be further cleaved to form a constitutively active, calmodulin-independent, 61-kD fragment. Microsequencing and amino acid analysis of purified peptides after proteolysis of the 61- and 64-kD fragments were used to determine the amino-terminal and carboxyl-terminal sequences of the 64-kD fragment. Cleavage within the calmodulin-binding region at Arg505 generates the catalytically inactive 64-kD fragment, which is incapable of binding calmodulin. Further digestion removes a carboxyl-terminal fragment, including the pseudosubstrate sequence Ser484-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met- Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg505 and results in a calmodulin-independent 61-kD fragment. Both the 61- and 64-kD fragments have the same primary amino-terminal sequences. These results provide direct support for the concept that the pseudosubstrate structure binds the active site and that the role of calmodulin is to modulate this interaction. Pseudosubstrates may be utilized in analogous ways by other allosterically regulated enzymes.

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“…Our knowledge of the primary structure of a nmMLCK, and the insight provided by our demonstration that the catalytic and CaM regulatory regions of nmMLCK and smMLCK are identical in amino acid sequence, allowed us to use the information gained from previous studies (19,23,48,50,78) of smMLCK to directly test, by site-specific mutagenesis, models (4, 27, 33, 39, 42-44, 48, 50, 61) of how MLCK structure might correlate with CaM binding and enzyme activation. All of the models have in common the assumption that there are key regions within the MLCK sequence that prevent the manifestation of protein kinase activity due to an intramolecular inhibition, or autoinhibition, and that activation by CaM results from the release of this intramolecular inhibition.…”

mentioning

confidence: 99%

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

Shoemaker

Lau

Shattuck

et al. 1990

The Journal of Cell Biology

165

114

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The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.

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Tryptic digestion of myosin light chain kinase produces an inactive fragment that is activated on continued digestion

Cited by 15 publications

References 6 publications

The magnesium-ATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme

The magnesium-ATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme

Autoregulation of Enzymes by Pseudosubstrate Prototopes: Myosin Light Chain Kinase

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

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