Allele frequencies for six PCR-based loci and three protein-based (i.e., enzyme systems) loci were determined in a Caucasian sample population from New Jersey. The loci are LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1, PGM1, ESD, and EAP. All loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the nine loci. The allelic frequency data generally are similar to another Caucasian population database.

In the last decade, as a result of molecular cloning and the reverse-transcriptase polymerase chain reaction, numerous isoforms of the contractile protein myosin have been discovered. What lags behind their discovery is knowledge of their functions. This review focuses on some of my recent work on the structure, function and regulation of isoforms of the heavy chain of vertebrate smooth muscle and nonmuscle myosin II. Reference to related work in the field is included where appropriate. The particular isoforms discussed are those that are generated by alternative splicing near the 5' end of the pre-mRNA, resulting in either an insertion or a deletion of a cassette of amino acids near the amino-terminus of the myosin heavy chain (MHC) protein. In both the smooth muscle and nonmuscle MHCs, this splicing occurs in the exact same region, which begins at amino acid 212 in the primary sequence. In the three-dimensional structure of the molecule, these inserts are located near the ATP-binding pocket in a region of the MHC that was not resolved in the crystal structure and therefore is believed to represent a flexible loop. In the smooth muscle MHC, the insertion of seven amino acids in this loop confers a higher enzymatic activity on the myosin. The potential mechanism by which this occurs and the significance to smooth muscle contractile diversity is discussed. In the nonmuscle MHC, the insert in this region is a different size and sequence of amino acids than that in the smooth muscle MHC. A serine residue (Ser-214) in the nonmuscle loop is phosphorylated by p34cdc2 kinase in Xenopus during meiotic maturation of oocytes to eggs and is dephosphorylated in interphase egg extracts that are equivalent to the interphase after fertilization of the egg. Thus, MHC-B phosphorylation by cdc2 kinase correlates with the cortical reorganization that occurs during meiosis, and dephosphorylation correlates with the cortical contraction that occurs at fertilization, which aids in pronuclear fusion. In summary, these inserts in the MHC molecule, in a flexible loop near the ATP-binding pocket, appear to be important in determining differences in function or regulation among myosin II isoforms.

An expressed gene formed by fusion between the CBFB transcription factor gene and the smooth muscle myosin heavy chain gene MYH11 is consistently detected by reverse transcription polymerase chain reaction (RT-PCR) in patients who have acute myeloid leukemia (AML) subtype M4Eo with an inversion of chromosome 16. We have previously shown that a CBFB-MYH11 cDNA construct can produce a chimeric protein and transform NIH 3T3 cells. However, the presence of the chimeric protein in patient cells has not been demonstrated previously. Here, we show that such chimeric proteins can be identified in vivo, primarily in the nuclei of the leukemic cells, by use of antibodies against the C-terminus of the smooth muscle myosin heavy chain and the fusion junction peptide. A very high molecular weight protein/DNA complex is generated when nuclear extracts from patient cells are used in electrophoretic mobility shift assays, as seen in NIH 3T3 cells transfected with the CBFB-MYH11 cDNA. Immunofluorescence staining shows that the proteins are organized in vivo into novel structures within cell nuclei. One isoform of the transcript of the CBFB-MYH11 fusion gene, containing the MHC204 C-terminus, was the predominant from in all five cases studied.

Protein kinase C-delta (PKC-delta) has been demonstrated to be phosphorylated on tyrosine residue(s) in many different biological systems (Li, W., Yu, J.-C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735; Li, W., Mischak, H., Yu, J.-C., Wang, L.-M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994) J. Biol. Chem. 269, 2349-2352; Denning, M. F., Dlugosz, A. A., Howett, M. A., and Yuspa, S. H. (1993) J. Biol. Chem. 268, 26079-26081). Tyrosine phosphorylation of PKC-delta has also been shown to occur in vitro when purified PKC-delta is coincubated with different tyrosine kinase sources. However, the tyrosine phosphorylation site(s) is currently unknown and the exact effect of this phosphorylation on its serine/threonine kinase activity and biological functions is still controversial. To directly investigate the potential role of PKC-delta tyrosine phosphorylation, tyrosine 187 was converted to phenylalanine (PKC-delta Y187F) by site-directed mutagenesis, and expression vectors containing PKC-delta Y187F cDNAs were transfected into both 32D myeloid progenitor cells and NIH 3T3 fibroblasts. The results showed that tyrosine 187 of PKC-delta became phosphorylated in vivo in response to 12-O-tetradecanoylphorbol-13-acetate stimulation or platelet-derived growth factor receptor activation. In vivo labeling and subsequent two-dimensional phosphopeptide analysis demonstrated that one phosphopeptide was absent in PKC-delta Y187F when compared to wild type PKC-delta, further substantiating that tyrosine 187 of PKC-delta is phosphorylated in vivo. Although the phosphotyrosine content of PKC-delta Y187F was reduced compared with PKC-delta WT, the kinase activity of PKC-delta Y187F toward a PKC-delta substrate was not altered. Moreover, 12-O-tetradecanoylphorbol-13-acetate-mediated monocytic differentiation of 32D cells was not affected by expression of the PKC-delta Y187F mutant. Taken together, these results suggest that tyrosine phosphorylation of PKC-delta on 187 may not influence PKC-delta activation and known functions.

An inversion of chromosome 16 associated with the M4Eo subtype of acute myeloid leukemia produces a chimeric protein fusing the beta subunit of the transcription factor core binding factor (CBF beta) to the tail region of smooth muscle myosin heavy chain (SMMHC). We investigated the oncogenic properties of this CBF beta-SMMHC chimeric protein using a 3T3 transformation assay. NIH 3T3 cells expressing CBF beta-SMMHC acquired a transformed phenotype, as indicated by their ability to form foci, grow in soft agarose, and form tumors in nude mice. Cells expressing normal CBF beta or the SMMHC tail domain did not become transformed. Electrophoretic mobility-shift assays showed that extracts from cells transformed by CBF beta-SMMHC no longer formed the normal CBF/DNA complex but instead formed a much larger complex that did not migrate into the gel. Analysis of CBF beta-SMMHC deletion mutants demonstrated that the chimeric protein was transforming only if two domains were both present: (i) CBF beta sequences necessary for association with the CBF alpha subunit, and (ii) SMMHC sequences important for the formation of multimeric filaments. These results are direct evidence that CBF beta-SMMHC can function as an oncoprotein.

In this paper we review some of our recent work on the structural and biochemical characterization of isoforms of the heavy chain of vertebrate smooth muscle myosin II. There exist both amino-terminal and carboxyl-terminal alternatively spliced isoforms of the smooth muscle myosin heavy chain (MHC). mRNA splicing at the 3' end generates two MHCs, which differ in length and amino acid sequence in the carboxyl terminus. We will refer to the longer, 204-kDa isoform as MHC(204) and the shorter, 200-kDa isoform as MHC(200). We found that MHC(204), but not MHC(200), can be phosphorylated by casein kinase II on a serine near the carboxyl terminus, suggesting that these isoforms may be differentially regulated. The physiological significance of this phosphorylation is not known. However, as demonstrated in this paper, phosphorylation does not appear to affect filament formation, velocity of movement of actin filaments by myosin in an in vitro motility assay, actin-activated Mg2+ ATPase activity, or myosin conformation. Our results also show that MHC(204) and MHC(200) form homodimers, but not heterodimers. Purified MHC(204) and MHC(200) homodimers are not enzymatically different, at least as measured using an in vitro motility assay. The amino-terminal spliced MHC(204) and MHC(200) isoforms are the result of the specific insertion or deletion of seven amino acids near the ATP-binding region in the myosin head. We refer to these isoforms as inserted (MHC(204)-I; MHC(200)-I) or noninserted (MHC(204); MHC(200)), respectively. In contrast to the carboxyl-terminal spliced isoforms, the amino-terminal spliced inserted and noninserted myosin heavy chain isoforms are enzymatically different. The inserted isoform, which is expressed in intestinal, phasic-type smooth muscle, has a higher actin-activated Mg ATPase activity and moves actin filaments at a greater velocity in an in vitro motility assay than the noninserted MHC isoform, which is expressed in tonic-type vascular smooth muscle. The results presented in this review suggest that the alternative splicing of smooth muscle mRNA results in at least four different isoforms of the myosin heavy chain molecule. The potential relevance of these molecular isoforms to smooth muscle function is discussed.

To identify new members of a family of protein-tyrosine phosphatases (PTPs), of which VH1 is prototype, we screened a B5/589 human mammary epithelial cell cDNA library by low stringency hybridization with probes for the catalytic domains of the human VHR and mouse 3CH134 phosphatases. Two overlapping clones of 1.8 and 2.5 kilobase pairs were detected by 3CH134 but not VHR probes. Sequence analysis of the largest clone, B23, revealed a 2470-nucleotide open reading frame encoding a novel protein. Within the 397 amino acid sequence, the HCXAGXXR signature sequence for PTPs was located at positions 261-268. The closest similarities were to 3CH134, its human homolog CL100, and PAC-1, PTPs induced as early response genes to mitogen stimulation. Less relatedness was observed with VHR and VH1 dual specificity phosphatases of human and vaccinia virus, respectively. A bacterially expressed recombinant protein containing the catalytic domain of B23 showed significant but consistently lower activity than VHR in vitro. Among the substrates tested, B23 displayed the highest relative activity toward phosphorylated extracellular signal regulated kinase-1, suggesting that it may be a target for B23 activity in vivo. The B23 transcript was detected in a wide variety of normal human tissues, with relatively high expression in pancreas and brain. B23 was induced by serum stimulation of human fibroblasts as well as by heat shock with similar kinetics to those observed with CL100. Thus, B23 is a new human protein phosphatase which appears to be regulated in response to mitogenic signaling and at least some forms of stress.

The molecular mechanisms underlying the heterogeneity in contractile properties observed among smooth muscle tissues are unknown. We examined whether part of this diversity might be intrinsic to myosin by comparing structural and enzymatic properties of myosins from two physiologically diverse tissues. Using the reverse transcriptase polymerase chain reaction, we compared avian intestinal smooth muscle and vascular smooth muscle myosin heavy chain (MHC) mRNA. We found that intestinal, but not vascular, MHC mRNA contains an insert of 21 nucleotides, encoding 7 amino acids, in a region near the ATP binding site in the myosin head. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified myosin revealed that the relative mobilities of the previously described intestinal MHC isoforms SM1 (204 kDa) and SM2 (200 kDa) were slower than the corresponding vascular SM1 and SM2 isoforms. Furthermore, antibodies raised against a synthetic peptide corresponding to the deduced amino acid sequence of the intestinal insert strongly recognized intestinal SM1 and SM2 but only weakly recognized the vascular isoforms. The presence of the insert in intestinal myosin correlated with a higher velocity of movement of actin filaments in vitro and a higher actin-activated Mg2+-ATPase activity, compared with vascular myosin. Other than the MHC insert, one other structural difference distinguished intestinal and vascular myosins: two isoforms of the 17-kDa myosin light chain were found in vascular myosin, whereas a single isoform was found in intestinal myosin. Exchange of the intestinal myosin light chains onto the vascular MHC did not alter its activity in the in vitro motility assay, suggesting that the 7-amino acid MHC insert is responsible for the different enzymatic activities of vascular and intestinal myosins.

In this article we review the various amino acids present in vertebrate nonmuscle and smooth muscle myosin that can undergo phosphorylation. The sites for phosphorylation in the 20 kD myosin light chain include serine-19 and threonine-18 which are substrates for myosin light chain kinase and serine-1 and/or -2 and threonine-9 which are substrates for protein kinase C. The sites in vertebrate smooth muscle and nonmuscle myosin heavy chains that can be phosphorylated by protein kinase C and casein kinase II are also summarized. Original data indicating that treatment of human T-lymphocytes (Jurkat cell line) with phorbol 12-myristate 13-acetate results in phosphorylation of both the 20 kD myosin light chain as well as the 200 kD myosin heavy chain is presented. We identified the amino acids phosphorylated in the human T-lymphocytes myosin light chains as serine-1 or serine-2 and in the myosin heavy chains as serine-1917 by 1-dimensional isoelectric focusing of tryptic phosphopeptides. Untreated T-lymphocytes contain phosphate in the serine-19 residue of the myosin light chain, and in a residue tentatively identified as serine-1944 in the myosin heavy chain.

Vertebrate smooth muscle myosin heavy chains (MHCs) exist as two isoforms with molecular masses of 204 and 200 kDa (MHC204 and MHC200) that are generated from a single gene by alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). A dimer of two MHCs associated with two pairs of myosin light chains forms a functional myosin molecule. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHC204 were generated and used to immunoprecipitate purified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. MHC204 quantitatively removed from this mixture was completely free of MHC200. Immunoprecipitation of the supernatant with an antiserum that recognizes both isoforms equally well revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. A method is described for the purification of enzymatically active MHC204 and MHC200 homodimers by affinity chromatography of myosin on a protein G-agarose high performance liquid chromatography column containing immobilized MHC204 antibodies. We show, using an in vitro motility assay, that the movement of actin filaments by myosin containing 204-kDa heavy chains (0.435 +/- 0.115-mu-m/s) was not significantly different from that of myosin containing 200-kDa heavy chains (0.361 +/- 0.078-mu-m/s) or from myosin containing equal amounts of each heavy chain isoform (0.347 +/- 0.082-mu-m/s).

The heavy chain of smooth muscle myosin was found to be phosphorylated following immunoprecipitation from cultured bovine aortic smooth muscle cells. Of a variety of serine/threonine kinases assayed, only casein kinase II and calcium/calmodulin-dependent protein kinase II phosphorylated the smooth muscle myosin heavy chain to a significant extent in vitro. Two-dimensional maps of tryptic peptides derived from heavy chains phosphorylated in cultured cells revealed one major and one minor phosphopeptide. Identical tryptic peptide maps were obtained from heavy chains phosphorylated in vitro with casein kinase II but not with calcium/calmodulin-dependent protein kinase II. Of note, the 204-kDa smooth muscle myosin heavy chain but not the 200-kDa heavy chain isoform was phosphorylated by casein kinase II. Partial sequence of the tryptic phosphopeptides generated following phosphorylation by casein kinase II yielded Val-Ile-Glu-Asn-Ala-Asp-Gly-Ser*-Glu-Glu-Glu-Val. The Ser* represents the Ser(PO4) which is in an acidic environment, as is typical for casein kinase II phosphorylation sites. By comparison with the deduced amino acid sequence for rabbit uterine smooth muscle myosin (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737), we have localized the phosphorylated serine residue to the non-helical tail of the 204-kDa isoform of the smooth muscle myosin heavy chain. The ability of the 204-kDa isoform, but not the 200-kDa isoform, to serve as a substrate for casein kinase II suggests that these two isoforms can be regulated differentially.