Snapping of the carboxyl terminal tail of the catalytic subunit of PKA onto its core: characterization of the sites by mutagenesis.

A set of 45 mutants of the carboxyl terminal tail of the PKA catalytic subunit was prepared and used to assess the contribution of this tail to the structure and function of the kinase. Ala substitutions of Asp 323, Phe 327, Glu 333, and Phe 350 resulted in a complete loss of enzymatic activity. Other replacements by Ala (Phe 314, Tyr 330, Glu 332, and Phe 347) brought about either a drop in activity to less than 10% of the wild-type enzyme or a reduction of affinity toward ATP (Lys 317, Lys 319, Tyr 330, and Glu 332) or toward Kemptide (Ile 315, Tyr 330, Val 337, Ile 339, Lys 345, and Glu 346). Mutations of Ser 338, a major autophosphorylation site of PKA, by Ala, Glu, Asp, Gln, and Asn showed that the kinetic parameters of these mutants are similar to those of the wild-type. The contribution of each of these tail mutations to the structure and stability of the kinase was assessed by monitoring its effect on the heat stability (when measurable) or by determining the susceptibility of the mutant kinase to cleavage by the Kinase Splitting Membranal Proteinase/Meprin beta. Here we show that the tail of PKA has a key role in creating the active conformation of the kinase. It does so by means of specific amino acid residues, which act as "snapping points" to embrace the two lobes of the kinase and orient them in the correct juxtaposition for substrate docking, biorecognition, and catalysis.

The negative charge of Glu-127 in protein kinase A and its biorecognition.

A set of mutants of protein kinase A (PKA) in which Gln-127 was replaced by Gln, Asp, Asn, and Arg was prepared. Their Km and Vmax values show that the negative charge of Glu-127 (not merely its hydrogen bonding capacity) is indispensable for the kinase activity, since Glu-127/Gln is inactive, in spite of the fact that it can form hydrogen bonds and is very similar in bulkiness and conformation to wt-PKA. Glu-127 is involved in the biorecognition of PKA, interacting ionically with the positively charged guanido group of Arg P-3 (a major recognition element in the consensus sequence of PKA). In support of this conclusion, it is shown that a regression of the Glu-127 carboxylate by 1.54 A (as in Glu-127/Asp) results in an active kinase with a similar thermal stability and susceptibility to conformation-dependent proteolysis, a similar Vmax, an identical Km for ATP, but a > 20-fold higher Km for kemptide. The two inactive mutants of PKA, Glu-127/Gln and Glu-127/Asn, are potentially useful for studying protein-protein interactions of PKA, e.g. for monitoring enzymatically the displacement of active PKA from its complexes.

Unveiling the substrate specificity of meprin beta on the basis of the site in protein kinase A cleaved by the kinase splitting membranal proteinase.

The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu332-Glu333, within the cluster of acidic amino acids (Asp328-Glu334) of the kinase. The Km values of KSMP and of meprin beta (which reproduces KSMP activity) for the C-subunit are below 1 microM. The Km for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin beta. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the Kcat of the proteinase. These studies unveil a new specificity for meprin beta, suggesting new substrates that are 1-2 orders of magnitude better in their Km and Kcat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin beta is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.

Anti-head and anti-tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A. Use in the study of the kinase splitting membranal proteinase KSMP.

Protein kinases share a considerable sequence homology in their catalytic core (residues 40-300 in PKA). Each core is flanked by "head" and "tail" segments at its amino- and carboxy-termini, which are different in the various kinases. These end segments may play an important role in creating the preferential affinity of each kinase for its physiological substrates or regulatory ligands. Here we describe three anti-peptide antibodies (alpha P-1, alpha P-2, and alpha P-3) that specifically recognize the head and tail segments of the catalytic subunit (C) of PKA. (i) alpha P-1 (against 6A-K23) react with C when denatured but not when in its native structure; (ii) alpha P-2 (against 319K-I335), bind to the site in C cleaved by the kinase splitting membranal proteinase (KSMP) and inhibit this cleavage of C; (iii) alpha P-3 (against 338S-F350) react with C but not with the KSMP cleavage product C', useful for detecting a KSMP-like activity in different tissues and subcellular loci. The combined use of the antibodies described here provides a strict definition of C, and thus a high degree of fidelity in its biorecognition.

Structural requirements for inducible shedding of the p55 tumor necrosis factor receptor.

Induced shedding of the p55 tumor necrosis factor receptor (p55-R) was previously shown to be independent of the amino acid sequence properties of the intracellular domain of this receptor. We now find it also independent of the sequence properties of the transmembrane domain and of the cysteine-rich region that constitutes most of the extracellular domain of the receptor. The shedding is shown to depend solely on the sequence properties of a small region within the spacer that links the cysteine-rich region in the extracellular domain to the transmembrane domain. Detailed tests of effects of mutations in the spacer on the shedding indicate that the process is independent of the amino acid side-chain identity in this region except for a limited dependence on the identity of 1 residue (Val-173), located downstream to the putative major cleavage site of the receptor. It is strongly affected, however, by some mutations that seem to change the conformation of the spacer region. These findings suggest that a short amino acid sequence in the p55-R is essential and sufficient for its shedding and that the shedding is mediated either by a protease with limited sequence specificity or by several different proteases that recognize different amino acid sequences, yet it strictly depends on some conformational features of the cleavage region in the receptor.