Different molecular mechanisms for Rho family GTPase-dependent, Ca2+-independent contraction of smooth muscle.

Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.

Tryptophan residues in caldesmon are major determinants for calmodulin binding.

Calmodulin has been shown to interact with the COOH-terminal domain of gizzard h-caldesmon at three sites, A (residues 658-666), B (residues 687-695), and B' (residues 717-725), each of which contains a Trp residue [Zhan et al. (1991) J. Biol. Chem. 266, 21810-21814; Marston et al. (1994) J. Biol. Chem. 296, 8134-8139; Mezgueldi et al. (1994) J. Biol. Chem. 269, 12824-12832]. To determine the contribution of each of the three Trp residues in the calmodulin-caldesmon interaction, we have mutated the Trp residues to Ala in the COOH-terminal domain of fibroblast caldesmon (CaD39) and studied the effects on calmodulin binding by fluorescence measurements and using immobilized calmodulin. Wild-type CaD39 binds with a Kd of 0.13 x 10(-6) M and a stoichiometry of 1 mol of calmodulin per mol of caldesmon. Replacing Trp 659 at site A or Trp 692 at site B to Ala reduces binding by 22- and 31-fold (Kd = 2.9 x 10(-6) and 4.0 x 10(-6) M), respectively, and destabilizes the CaD39-calmodulin complex by 1.75 and 1.94 kcal mol-1, respectively. Mutation of both Trp 659 and Trp 692 to Ala further reduces binding with a Kd of 6.1 x 10(-6) M and destabilizes the complex by 2.17 kcal mol-1. On the other hand, mutation of Trp 722 at site B' to Ala causes a much smaller decrease in affinity (Kd = 0.6 x 10(-6) M) and results in a destabilization energy of 0.87 kcal mol-1. To investigate the relative importance of the amino acid residues near each Trp residue in the caldesmon-calmodulin interaction, deletion mutants were constructed lacking site A, site B, and site A + B. Although deletion of site A decreases binding of CaD39 to calmodulin by 13-fold (Kd = 1.7 x 10(-6) M), it results in tighter binding than mutation of Trp 659 to Ala at this site, suggesting that the residues neighboring Trp 659 may contribute negatively to the interaction. Deletion of site B causes a similar reduction in binding (Kd = 4.1 x 10(-6) M) as observed for replacing Trp 692 to Ala at this site, indicating that Trp 692 is the major, if not the only, binding determinant at site B. Deletion of both site A and site B drastically reduces binding by 62-fold. Taken together, these results suggest that Trp 659 and Trp 692 are the major determinants in the caldesmon-calmodulin interaction and that Trp 722 in site B' plays a minor role.

A germ line-specific sequence element in an intron in Tetrahymena thermophila.

In the ciliated protozoa, the germ line micronucleus carries DNA sequences that are eliminated during the developmental program that produces the somatic macronucleus. We have identified such a micronuclear element in an intron of a gene in the holotrichous ciliate, Tetrahymena thermophila. The macronuclear version of this gene, numbered from the transcription initiation site to the polyadenylation site, is 1569 nucleotides long and contains two introns: intron 1 from nucleotides 206 to 382 and intron 2 from nucleotides 665 to 898. The gene is transcribed into a stable polyadenylated mRNA which is predicted to encode a polypeptide with a highly repetitive structure and an unknown function. The micronuclear version of the gene contains a 2.9-kb sequence element, mse2.9, in intron 2. This element was characterized by sequencing two micronuclear genomic clones that contain 909 and 764 base pairs of micronucleus-specific sequence from its left and right ends, respectively. This sequence has an (A+T) content of 81% and is present as a single copy. The termini of mse2.9 are located within a 4-nucleotide direct repeat, TTAT, one copy of which is retained in the macronuclear gene, between positions 755 and 759. Other features near the termini of mse2.9 include a GC-rich sequence motif, CCAACCCGTTG, 31 nucleotides outside the left terminus and a 20-nucleotide palindrome 6 nucleotides outside the right terminus.

Sequence heterogeneity in the duplicate large subunit ribosomal RNA genes of Tetrahymena pyriformis mitochondrial DNA.

In the ciliated protozoan, Tetrahymena pyriformis, the mitochondrial large subunit ribosomal RNA (LSU rRNA) is discontinuous, consisting of two discrete RNA species: a 280-nucleotide LSU alpha (constituting the 5'-portion) and a 2315-nucleotide LSU beta (corresponding to the remaining 3'-portion of this rRNA). The T. pyriformis mitochondrial genome contains two copies of the LSU alpha.beta gene complex, and we have previously provided evidence that both copies are transcribed (Heinonen, T. Y. K., Schnare, M. N., Young, P. G., and Gray, M. W. (1987) J. Biol. Chem. 262, 2879-2887). We now report the complete sequences of the two copies of the LSU alpha.beta gene complex. These are not identical, but differ at 5 out of the 2595 positions by single nucleotide substitutions in one sequence relative to the other. In the secondary structure model we propose here, two of these differences are located in base-paired regions of the LSU rRNA; however, they do not interrupt the complementary interactions in these helices. The other three differences occur in single-stranded regions of the secondary structure. The base substitutions documented here are not localized to those regions of LSU rRNA that are the most highly conserved in global phylogenetic comparisons, and therefore it seems unlikely that they are of fundamental functional significance. Whether they might exert more subtle effects on ribosome function remains to be determined.

Rearranged coding segments, separated by a transfer RNA gene, specify the two parts of a discontinuous large subunit ribosomal RNA in Tetrahymena pyriformis mitochondria.

In the mitochondria of Tetrahymena pyriformis ST, the large subunit ribosomal RNA (LSU rRNA) is discontinuous, consisting of two species, alpha (280 nucleotides in length) and beta (approximately equal to 2.45 kilobases long). LSU alpha is the 5'-terminal portion (i.e. a 5.8 S-like rRNA), and LSU beta constitutes the rest of this LSU rRNA, as judged by both primary and secondary structure homology to other LSU rRNAs. Remarkably, LSU alpha is encoded downstream of LSU beta, the two genes being separated by a tRNALeu gene. This is the first demonstration of a discontinuous rRNA whose coding sequences are rearranged, deviating from the conventional, highly conserved, 5'----3' order of sequence domains in the LSU rRNA gene. This novel gene organization (5'-LSU beta-2-base pair spacer-tRNALeu-10-base pair spacer-LSU alpha) is identical in both copies of the subterminal inverted repeat in the linear T. pyriformis mitochondrial genome. Sequence heterogeneity in the LSU alpha transcript and its genes, together with Northern hybridization data, suggest that both copies of the inverted repeat are expressed.

A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria.

We show here that in the mitochondria of Tetrahymena pyriformis, the small subunit (SSU) rRNA is discontinuous, being comprised of two separate components which we term "alpha" (a novel low molecular weight RNA, approximately equal to 200 nucleotides long) and "beta" (a previously described 14 S RNA). The SSU alpha rRNA has been sequenced in its entirety; it represents the immediate 5'-terminal domain of conventional SSU rRNA. The sequences at the ends of the SSU beta rRNA have also been determined; they show that this molecule corresponds to the 3'-terminal 7/8 of conventional SSU rRNA. A 2.5-kilobase pair XbaI restriction fragment of T. pyriformis mitochondrial DNA which contains the SSU alpha and SSU beta rRNA genes was cloned and its complete nucleotide sequence was determined. This revealed that the genes encoding the two segments of SSU rRNA are separated by a 54-base pair (A + T)-rich spacer. The alpha and beta sequences can be fitted to a generalized secondary structure model for eubacterial 16 S rRNA, with the two RNA species associating through long range interactions to form base-paired regions characteristic of SSU rRNA. In this model, the spacer is situated in a region of pronounced primary and secondary structural variation among SSU rRNAs. The significance of these findings with respect to rRNA biosynthesis and processing and the possible evolutionary relationship between spacers and variable regions in rRNA genes is discussed.

Phenylalanine and tyrosine transfer RNAs encoded by Tetrahymena pyriformis mitochondrial DNA: primary sequence, post-transcriptional modifications, and gene localization.

We have isolated Phe and Tyr tRNAs from Tetrahymena pyriformis mitochondria and have determined that these are "native" species, encoded by the mtDNA. A single gene for the tRNA(Phe) has been positioned 12-14 kbp from the left end of the linear Tetrahymena mtDNA, while duplicate tRNA(Tyr) genes have been localized within the inverted terminal repeats of this genome. Primary sequence analysis demonstrates that the tRNA(Tyr) has all of the characteristic primary and secondary structural features of a normal tRNA; however, the tRNA(Phe) displays several atypical features, including (i) replacement of the usual T psi sequence by UC, (ii) a U.U pair in the T psi C stem, and (iii) an extra 5'-nucleotide (U).