Bruce E. Kemp

Comparison of the effects of amino-terminal synthetic parathyroid hormone-related peptide (PTHrP) of malignancy and parathyroid hormone on resorption of cultured fetal rat long bones

SummaryWe have compared the effects of of various synthetic amino-terminal forms of human parathyroid hormone-related peptide (PTHrP) of malignancy with synthetic parathyroid hormone (PTH) on the resorptive responses of fetal rat long bones in organ culture. PTH and PTHrP increased45Ca release at concentrations of 0.1–25 nM. PTHrP (1–40) and bovine PTH (1–34) were more potent than human PTH (1–34) and PTHrP (1–34). However, the slopes of the dose-response curves and the maximal resorptive effects were similar. There was a marked decrease in the potency of amino-terminal PTHrP peptides as the length was decreased. PTHrP (1–29) and PTHrP (1–25) were inactive at 120 nM. Further comparison of bPTH (1–34) and PTHrP (1–34) showed that both could induce bone resorption after a brief (6 hours) exposure and that the response to PTHrP (1–34) was qualitatively similar to that of bPTH (1–34) with respect to enhancement by ACTH and inhibition by calcitonin and glucocorticoids. Hydroxyurea and indomethacin did not block the resorptive response to either agonist. Cyclic AMP production in response to PTHrP (1–34) and (1–40) was similar to that for bPTH (1–34) in ROS 17/2.8 cells. The cyclic AMP (cAMP) response was much smaller in fetal rat long bones and calvariae, and bPTH was more potent than PTHrP. These studies confirm that PTHrP is quantitatively similar in its effects on bone resorption to PTH and are consistent with the two agents acting on the same receptor.

Phosphorylation of ribosomal protein S6 and a peptide analogue of S6 by a protease-activated kinase isolated from rat liver

A trypsin‐activated protein kinase has been isolated from rat liver using a peptide analogue of ribosomal protein S6 as a substrate in kinase assays. The structure of the peptide, Arg‐Arg‐Leu‐Ser‐Ser‐Leu‐Arg‐Ala, was based on a region of S6 containing both an insulin‐ and cyclic AMP‐regulated phosphorylation site. The trypsin‐activated protein kinase phosphorylated a corresponding site in the peptide analogue and ribosomal protein S6 that was distinct from the preferred site for cyclic AMP‐dependent protein kinase. Ribosomal S6 contained at least one other major site for the trypsin‐activated protein kinase.

Recognition of envelope and tat protein synthetic peptide analogs by HIV positive sera or plasma

A series of synthetic peptides corresponding to segments of HIV encoded proteins were selected using criteria described by Welling et al. [(1985) FEBS Lett. 188, 215]. Synthetic peptide analogs to gpl20 (2–13), (55–65), gp41 (582–596) (659–670) and tatIII (71–83) were recognized by 41–67% of sera or plasma from individuals known to be infected with HIV on the basis of virus isolation or Western blot screening. The peptide which reacted with most sera or plasma was gp41 (582–596), a conserved region in the transmembrane glycoprotein. An extended peptide analog, gp41 (579–599), tested against the same samples showed almost 100% reactivity, confirming independent studies identifying a highly immunodominant region of gp41. There was an unexpected high prevalence of antibodies (52%) to the tatIII peptide.

Evidence for a second phosphorylation site on eIF-2α from rabbit reticulocytes

Ser 51 in the NH2‐terminal sequence of the α‐subunit of eukaryotic peptide initiation factor 2 (eIF‐2) has been identified as a second phosphorylation site for the heme‐controlled eIF‐2α kinase from rabbit reticulocytes. Increased phosphorylation of this serine relative to the previously described phosphorylation site (Ser 48) is observed when the kinase reaction is carried out in the presence of the α‐subunit of spectrin. A synthetic peptide corresponding to eIF‐2α(41–54) is phosphorylated only in Ser 51 by the eIF‐2α kinase.

Regulation of Smooth Muscle Myosin Light Chain Kinase by Calmodulin

The mutagenesis work described in this paper has been instrumental in furthering our understanding of how CaM binds to and activates MLCK. Figure 2 schematically represents this interaction. The inactive MLCK appears to have a catalytic domain that is repressed by a substrate inhibitory domain that overlaps with the CaM binding domain, a basic amphipathic helix. In the presence of Ca2+, CaM undergoes a conformational change that exposes two hydrophobic pockets, one in each globular lobe, that are important for binding to MLCK. Upon binding CaM, MLCK undergoes a conformational change that derepresses the catalytic site, allows substrate access and light chain phosphorylation. Calmodulin antagonist drugs intercalate within these hydrophobic pockets to interfere with target enzyme binding. The total loss of activity if W800 is altered to A illustrates the importance of these hydrophobic interactions within the enzyme. The basic residues are also important; most of the basic residues in the binding domain of MLCK appear to aid in CaM binding but are not in themselves crucial, this includes the RRK triad. However, a specific electrostatic interaction between R812 of MLCK and CaM is suggested by the complete failure in MLCK activation if this residue is changed to an A. Electrostatic interactions between MLCK and CaM are also indicated by the TaM-BM1 mutant. This mutant can bind to but not activate MLCK. It is hypothesized that TaM-BM1 will bind to the basic amphipathic helix of MLCK but that the alterations in the surface charges (especially E14 and T34) and/or hydrophobicity (S38) prevent the proper conformational change in MLCK necessary for light chain phosphorylation. The resulting MLCK-CaM complex is therefore, inactive but can bind TaM-BM1. The exact interaction of these amino acids in CaM with MLCK will have to await the elucidation of a CaM-MLCK co-crystal.

Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.

Hydroxyamino acid specificity of smooth muscle myosin light chain kinase

Synthetic peptides corresponding to the phosphorylation site in the myosin regulatory light chain from smooth muscle, Lys-Lys-Arg-Ala-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ala ([Ala14,15]MLC(11-23] and containing a variety of hydroxyamino acid analogs at position 19, were tested as substrates for the smooth muscle myosin light chain kinase. Peptide analogs containing either D-serine or cis-hydroxyproline were not phosphorylated. The corresponding trans-hydroxyproline containing peptide was poorly phosphorylated with a Km of 2.3 microM and a Vmax of 3 X 10(-3) mumol.min-1.mg-1 compared to a Km of 12.5 microM and a Vmax of 1.43 mumol.min-1.mg-1 for the parent peptide. All three hydroxyamino acid analog peptides acted as relatively potent inhibitors of myosin light chain phosphorylation with Ki values in the range 7.5-10 microM, comparable to 7 microM for the parent peptide. Thus the failure of the hydroxyamino acid analog peptides to act as effective substrates was not the result of poor binding to the enzyme. In contrast, the same substitutions made in the peptide substrate for the cAMP-dependent protein kinase resulted in poor inhibitors. It is likely that the hydroxyl group of the substituting amino acids in the myosin light chain peptide analogs is not presented in the correct orientation in the active site for transfer of the phosphate group.

Chemical modification of lysine and arginine residues in the myosin regulatory light chain inhibits phosphorylation

The contribution of lysine and arginine residues to the substrate specificity of the myosin light-chain kinase has been studied using chemically modified myosin light chains. Succinylation or maleylation of the myosin light chains caused complete inhibition of their phosphorylation. Modification of 50% of the lysine residues resulted in 90% inhibition of phosphorylation and this was accompanied by a 25-fold increase in the apparent Km. In contrast, phosphorylation of the myosin light chains by the cAMP-dependent protein kinase was relatively insensitive to lysine modification, with only a 15% reduction in phosphorylation following succinylation of 50% of the lysine residues. Treatment with either cyclohexane-1,2-dione or camphorquinone-10-sulfonic acid resulted in between 90 and 98% inhibition of myosin light-chain phosphorylation. These reagents caused modification of both lysine and arginine residues, and accordingly only part of the inhibition can be attributed to arginine modification. Modification of all of the cysteine and methionine residues caused only a 40% inhibition of phosphorylation. The results of this study support the concept that lysine and arginine residues act as essential specificity determinants for the myosin light-chain kinase in protein substrates.

Identification of Functional Domains of the Inhibitor Protein of Camp-Dependent Protein Kinase

Phosphorylation-dephosphorylation of proteins is now recognized as a dynamic regulatory process in many cellular functions. cAMP-dependent protein kinase is one of the major enzymes involved in this process. The kinase mediates most, if not all, of the cellular functions of cAMP. Physiologically, the series of events leading to cAMP-dependent protein phosphorylation begins with the binding of a hormone or other agent to a cell surface receptor. In many cases, the binding leads to the activation of adenylate cyclase and a consequential increase in the intracellular level of cAMP. In eukaryotes, the major, if not sole, cellular receptor of cAMP is the regulatory subunit of cAMP-dependent protein kinase. The kinase is activated upon binding of cAMP to its regulatory subunits. The activated kinase then phosphorylates cellular proteins and such modifications lead to changes in cellulat functions.