Mechanism of2-Chloro-(.epsilon.-amino-Lys75)-[6-[4-(N,N-diethylamino)phenyl]-1,3,5-triazin-4-yl]calmodulin Interactions with Smooth Muscle Myosin Light Chain Kinase and Derived Peptides

Protein kinases phosphorylate the appropriate protein substrate by recognizing residues both proximal and distal to the site of phosphorylation. Although these distal contacts may provide excellent binding affinities (low K m values) through stabilization of the enzymesubstrate complex, these contacts could reduce catalytic turnover (decrease k cat ) through slow phosphoprotein release. To investigate how protein kinases can overcome this problem and maintain both high substrate affinities and high turnover rates, the phosphorylation of the yeast RNA transport protein Npl3 by its natural protein kinase, Sky1p, was evaluated. Sky1p bound and phosphorylated Npl3 with a K m that was 2 orders of magnitude lower than a short peptide mimic representing the phosphorylation site and only proximal determinants. Surprisingly, this extraordinary difference is not the result of high affinity Npl3 binding. Rather, Npl3 achieves a low K m through a rapid and favorable phosphoryl transfer step. This step serves as a chemical clamp that locks the protein substrate in the active site without unduly stabilizing the product phosphoprotein and slowing its release. The chemical clamping mechanism offers an efficient means whereby a protein kinase can simultaneously achieve both high turnover and good substrate binding properties.The phosphorylation of hydroxyl-containing amino acids (i.e. serine, threonine, and tyrosine) in eukaryotic proteins controls numerous physiological responses essential for cell function. The enzymes that catalyze this modification, the protein kinases, have been studied for their critical role in complex signaling cascades and for their potential as valuable chemotherapeutic targets in proliferative diseases (1, 2). Although much is known about the protein kinase family on the structural and functional levels (3-6), less is known about the interactions of these enzymes with their physiological protein substrates. Protein kinases recognize and phosphorylate short peptide segments (consensus sequences) of 4 -10 residues based on the known phosphorylation sequences of their targets (7-9). Although these short segments (consensus sequences) are minimal substrates, they do not provide all the binding energy offered by the full-length protein substrate. For instance, the catalytic efficiency of phosphorylating the protein substrate MAPKAP2 using p38 MAPK 1 is ϳ2 orders of magnitude higher than that for a 14-residue peptide based on the consensus sequence (10). In another example, Csk phosphorylates the protein substrate Lck Ͼ3 orders of magnitude more efficiently than a 9-residue peptide based on the phosphorylation site (11). Studies of this type support a model in which regions beyond the limited consensus sequence interact with the protein kinase scaffold and govern high efficiency protein phosphorylation.Although an x-ray structure for a protein kinase with a full-length protein substrate bound is currently unavailable, structural and functional studies are now beginning to reveal the nature of the interactin...

Section: Dissociation Rate Constant For Mant-adp: Stopped-flow Fluorementioning

confidence: 89%

Chemical Clamping Allows for Efficient Phosphorylation of the RNA Carrier Protein Npl3

Aubol

Ungs

Lukasiewicz³

et al. 2004

“…The same workers also measured the corresponding rates for a 17-residue peptide whose sequence was taken from the calmodulin-binding domain of myosin light chain kinase. For this peptide, k 1 was more than 13,000 times faster at 5.3 ϫ 10 10 M Ϫ1 min Ϫ1 and k -1 was too slow to measure, being less than 0.6 min Ϫ1 (16). Comparison of the myosin light chain kinase peptide with the intact enzyme indicates that occlusion of the calmodulinbinding domain plays an important role in determining both the rate at which calmodulin binds and its affinity for the enzyme.…”

Section: Slow Activation Of Pmca By Calmodulinmentioning

confidence: 95%

“…For unphosphorylated Ca 2ϩ -calmodulin-dependent protein kinase, k 1 was about 600 times faster at 2.4 ϫ 10 9 M Ϫ1 min Ϫ1 and k -1 was 130 min Ϫ1 (15). For myosin light chain kinase, k 1 was about 1700 times faster at 6.6 ϫ 10 9 M Ϫ1 min Ϫ1 and k -1 was 34 min Ϫ1 (16). The same workers also measured the corresponding rates for a 17-residue peptide whose sequence was taken from the calmodulin-binding domain of myosin light chain kinase.…”

Section: Slow Activation Of Pmca By Calmodulinmentioning

confidence: 99%

The Rate of Activation by Calmodulin of Isoform 4 of the Plasma Membrane Ca2+ Pump Is Slow and Is Changed by Alternative Splicing

Caride¹,

Elwess²,

Verma³

et al. 1999

A reconstitution system allowed us to measure the ATPase activity of specific isoforms of the plasma membrane Ca 2؉ pump continuously, and to measure the effects of adding or removing calmodulin. The rate of activation by calmodulin of isoform 4b was found to be very slow, with a half-time (at 235 nM calmodulin and 0.5 M free Ca 2؉ ) of about 1 min. The rate of inactivation of isoform 4b when calmodulin was removed was even slower, with a half-time of about 20 min. Isoform 4a has a lower apparent affinity for calmodulin than 4b, but its activation rate was surprisingly faster (half time about 20 s). This was coupled with a much faster inactivation rate, consistent with its low affinity. A truncated mutant of isoform 4b also had a more rapid activation rate, indicating that the downstream inhibitory region of fulllength 4b contributed to its slow activation. The results indicate that the slow activation is due to occlusion of the calmodulin-binding domain of 4b, caused by its strong interaction with the catalytic core. Since the activation of 4b occurs on a time scale comparable to that of many Ca 2؉ spikes, this phenomenon is important to the function of the pump in living cells. The slow response of 4b indicates that this isoform may be the appropriate one for cells which respond slowly to Ca 2؉ signals.Unlike other mechanisms for removing Ca 2ϩ from the cytosol, the plasma membrane Ca 2ϩ pump requires activation by another protein, calmodulin. The requirement for this extra step may have profound effects on the shape of Ca 2ϩ spikes, particularly if the binding of calmodulin to the pump is slow. The activation by calmodulin of several calmodulin-regulated enzymes is fast, but it has been observed that the activation of the plasma membrane Ca 2ϩ pump is slow in human erythrocytes (1). Since the binding of calmodulin to the plasma membrane Ca 2ϩ pump is very tight, this slowness in the activation was surprising. Erythrocytes contain a mixture of isoforms 1 and 4 of the plasma membrane Ca 2ϩ pump (2), so it was not clear which isoform was responsible for the slow activation. It was possible to study the rate of activation in erythrocytes because almost all of their Ca 2ϩ -stimulated ATPase activity is due to the plasma membrane Ca 2ϩ pump, but extension of such studies to other cell types has been difficult. The major difficulty is the presence in almost all kinds of cells of non-pump Ca 2ϩ ATPases whose activity swamps that of the pump. Some studies using Ca 2ϩ indicators in whole cells other than erythrocytes have given results consistent with slow activation of the pump. In human neutrophils (3) it was concluded that a Ca 2ϩ spike was caused by delayed activation of the plasma membrane Ca 2ϩ pump. In this case the arguments were based in part on the use of a calmodulin antagonist, which is rather nonspecific. In vascular endothelial cells (4) a similar conclusion was based on the use of La 3ϩ , VO 4 3Ϫ and Hg 2ϩ as inhibitors of the plasma membrane Ca 2ϩ pump. Each of these reagents also inhibits other pumps a...

“…These In the two known cases, calmodulin has about a 25-40-fold higher affinity for the peptide than for the full-length enzyme. These cases used peptides representing the calmodulin-binding domains of myosin light chain kinase (28) and PMCA (16). The K d for the myosin light chain kinase peptide is 0.006 nM, whereas that for full-length myosin light chain kinase is 0.10 -0.22 nM.…”

Section: Discussionmentioning

confidence: 99%

Plasma Membrane Ca2+ Pump Isoform 3f Is Weakly Stimulated by Calmodulin

Filoteo¹,

Enyedi²,

Verma³

et al. 2000

Isoform 3f of the plasma membrane Ca2؉ pump is a major isoform of this pump in rat skeletal muscle. It has an unusual structure, with a short carboxyl-terminal regulatory region of only 33 residues when compared with the 77 to 124 residues found in the other isoforms. Also, whereas the regulatory regions of the other isoforms, downstream of the alternative splice, consist of two homologous groups, the sequence of 3f is not related to either group. A synthetic peptide representing the calmodulin binding domain of isoform 3f had a much lower calmodulin affinity (with a K d of 15 nM) than the corresponding peptide of isoform 2b (K d value was 0.2 nM). The characteristics of this domain were further studied by making chimeras of the 3f regulatory region with the catalytic core of isoform 4 and by making the full-length isoform 3f. Both constructs bound to calmodulin-Sepharose. The chimera was fully active without calmodulin, showing no stimulation of activity when calmodulin was added. The full-length isoform 3f was slightly activated by calmodulin. These data show that the regulatory region of isoform 3f is only a weak autoinhibitor of the enzyme, in contrast to the properties of all the other isoforms studied so far. Rather, this isoform is a special-purpose, constitutively active form of the enzyme, expressed primarily in skeletal muscle and as a minor isoform in brain.