Phosphorylation of human tau protein by microtubule-associated kinases: GSK3beta and cdk5 are key participants.

Microtubules (MTs), primarily composed of alpha and beta tubulin polymers, must often work in concert with microtubule-associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the "normal" physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimer's disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule-associated kinases most readily impacts the phosphorylation of tau at "AD-like" sites. Results from this study indicate that PKA, CK1, GSK3beta, and cdk5 associate with microtubules. Among the MT-associated kinases, GSK3beta and cdk5 most readily contribute to the ATP-induced "AD-like" phosphorylation of tau.

Characterization of microtubule-associated proteins in teleosts.

Although microtubules are known to play an important role in many cellular processes, they have been virtually neglected in fish. In this report, microtubule-associated proteins (MAPs) in fish (teleost) were characterized using antibodies (Abs) directed against the mammalian MAPs tau, MAP1A and B, and MAP 2. Two different populations of tau-like proteins (TLPs) were found in fish brain using the anti-tau Abs Tau-1, Tau-2, tau5', and tau3'. The TLPs that were recognized by Tau-1, Tau-2, and tau5' were (1) heat-stable; (2) the same molecular weight as mammalian TLPs: 59-62 kDa; (3) not enriched in microtubules prepared from catfish brain; and (4) localized to the cell body of neurons in fish brains. While the TLPs recognized by tau3' Abs were (1) heat-stable; (2) lower molecular weight than mammalian TLPs: 32-55 vs. 50-65 kDa; (3) enriched in microtubule fractions prepared from catfish brain, and (4) localized to the axons of neurons. These results are consistent with two different populations of TLPs being present in fish brains. While MAP2 was found to be approximately the same molecular weight, 250 kDa, in zebrafish and goldfish as in mammals and to be distributed to dendrites in the fish brain, both MAP1A and MAP1B were found to be about 25% the mass of their mammalian homologs. These results suggest that MAPS in fish have some characteristics similar to their mammalian counterparts, but also possess some unique properties that require further study to elucidate their function.

Sequence analysis of the Escherichia coli dnaE gene.

We have determined the sequence of a 4,350-nucleotide region of the Escherichia coli chromosome that contains dnaE, the structural gene for the alpha subunit of DNA polymerase III holoenzyme. The dnaE gene appeared to be part of an operon containing at least three other genes: 5'-lpxB-ORF23-dnaE-ORF37-3' (ORF, open reading frame). The lpxB gene encodes lipid A disaccharide synthase, an enzyme essential for cell growth and division (M. Nishijima, C.E. Bulawa, and C.R.H. Raetz, J. Bacteriol. 145:113-121, 1981). The termination codons of lpxB and ORF23 overlapped the initiation codons of ORF23 and dnaE, respectively, suggesting translational coupling. No rho-independent transcription termination sequences were observed. A potential internal transcriptional promoter was found preceding dnaE. Deletion of the -35 region of this promoter abolished dnaE expression in plasmids lacking additional upstream sequences. From the deduced amino acid sequence, alpha had a molecular weight of 129,920 and an isoelectric point of 4.93 for the denatured protein. ORF23 encoded a more basic protein (pI 7.11) with a molecular weight of 23,228. In the accompanying paper (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987), the sequence of the upstream region that contains lpxA and lpxB is reported.

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Biochemical characterization of a 34-kilodalton normal cellular substrate of pp60v-src and an associated 6-kilodalton protein.

Transformation of fibroblasts by several retroviruses that produce transforming gene products associated with protein kinase activity results in the phosphorylation of a normal cellular protein with an Mr of 34,000 (the 34K protein). Evidence is presented here that, as extracted from chicken embryo fibroblasts, this protein exists in two forms that differ both in their elution from hydroxylapatite and in their native molecular weight. The form that eluted from hydroxylapatite at 210 to 295 mM potassium phosphate displayed a native molecular weight of 30,000 to 40,000, whereas the form that eluted at 320 to 440 mM displayed a native molecular weight of 60,000 to 70,000. The latter form copurified with a low-molecular-weight protein with an approximate Mr of 6,000 (6K). Both forms of 34K were completely separable from malate dehydrogenase activity. Phosphorylated 34K, isolated from Rous sarcoma virus-transformed cells, was also present in two forms; hence, in the cell neither form serves as a preferential substrate for pp60v-src. We found that the expression of 34K differed greatly in various avian tissues. In particular, it was present in the highest concentration in cultured fibroblasts and in very low concentration in brain tissue. Its expression in this tissue seems to be controlled at the level of transcription, since 34K mRNA in brain tissue was barely detectable. The expression of 6K was similar to that of 34K.