The sequence of the alternatively spliced sixth exon of alpha-tropomyosin is critical for cooperative actin binding but not for interaction with troponin.

Tropomyosins, a family of highly conserved coiled-coil actin binding proteins, can differ as a consequence of alternative expression of several exons (Lees-Miller, J., and Helfman, D. (1991) BioEssays 13, 429-437). Exon 6, which encodes residues 189-213 in long, 284-residue tropomyosins, has two alternative forms, exon 6a or 6b, both highly conserved throughout evolution. In alpha-tropomyosin, exon 6a or 6b is not specific to any one of the nine isoforms. Exon 6b encodes part of a putative Ca2+-sensitive troponin binding site in striated muscle tropomyosins, suggesting that the exon 6-encoded region may be specialized for certain tropomyosin functions. A series of recombinant, unacetylated tropomyosin exon 6 deletion and substitution mutants and chimeras was expressed in Escherichia coli to determine the requirements of exon 6 for tropomyosin function. Functional properties of the tropomyosins were defined by actin affinity measured by cosedimentation, troponin T affinity using a newly developed biosensor assay, and regulation of the actomyosin MgATPase. The region of tropomyosin encoded by exon 6 affects actin affinity but not thin filament assembly, troponin T binding, or regulation with troponin. The tropomyosins with exon 6a or 6b function normally whether a striated muscle exon 9a or smooth/non-muscle exon 9d is present. However, the effect of deleting 21 amino acids encoded by exon 6 or replacing it with a GCN4 leucine zipper sequence depends on the COOH-terminal sequence.

Mapping the functional domains within the carboxyl terminus of alpha-tropomyosin encoded by the alternatively spliced ninth exon.

Tropomyosins are highly conserved, coiled-coil actin binding proteins found in most eukaryotic cells. Striated and smooth muscle alpha-tropomyosins differ by the regions encoded by exons 2 and 9. Unacetylated smooth tropomyosin expressed in Escherichia coli binds actin with high affinity, whereas unacetylated striated tropomyosin requires troponin, found only in striated muscle, for strong actin binding. The residues encoded by exon 9 cause these differences (Cho, Y.-J., and Hitchcock-DeGregori, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10153-10157). We mapped the functional domains encoded by the alpha-tropomyosin exon 9a (striated muscle-specific) and 9d (constitutively expressed), by measuring actin binding and regulation of the actomyosin MgATPase by tropomyosin exon 9 chimeras and truncation mutants expressed in E. coli. We have shown that: 1) the carboxyl-terminal nine residues define the actin affinity of unacetylated tropomyosin; 2) in the presence of Ca2+, the entire exon 9a is required for troponin to promote fully high affinity actin binding; 3) the first 18 residues encoded by exon 9a are critical for the interaction of troponin with tropomyosin on the thin filament, even in the absence of Ca2+. The results give new insight into the structural requirements of tropomyosin for thin filament assembly and regulatory function.

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.

Calmodulin-dependent multifunctional protein kinase in Aspergillus nidulans.

A Ca2+/calmodulin (CaM)-dependent multifunctional protein kinase has been isolated from Aspergillus nidulans and purified to homogeneity. Unlike any CaM-dependent multifunctional protein kinase described previously, the native enzyme from Aspergillus behaves as a monomer. The calculated molecular weight is 41,200. NaDodSO4/PAGE reveals a single protein band with an apparent Mr of 51,000. Two-dimensional isoelectric focusing/NaDodSO4/PAGE of the purified enzyme showed one major and one minor more acidic Coomassie blue-stained spot, both of which bind 125I-labeled calmodulin in a calcium-dependent manner. The kinase is autophosphorylated in a calcium- and CaM-dependent manner, yielding an increase in the amount and number of more acidic forms of the enzyme. The Aspergillus kinase catalyzes the Ca2+/CaM-dependent phosphorylation of known substrates of type II Ca2+/CaM-dependent protein kinases, including glycogen synthase, microtubule-associated protein 2, synapsin, tubulin, gizzard myosin light chain, and casein. Cross-reactivity between antiserum raised against native rat brain protein kinase II and 125I-labeled Aspergillus kinase has been detected. Two forms of CaM have been isolated from Aspergillus nidulans, both of which activate the Aspergillus kinase at lower concentrations than that required for activation by bovine brain CaM.