Tubulin-binding Domain Research Articles

Identification of a tubulin binding motif on the P2X 2 receptor

To isolate proteins interacting with P2X receptors, GST fusion proteins containing the intracellular C terminal tail of P2X 2, P2X 5, or P2X 7 were used as bait to screen detergent extracts of rat brain synaptosomes. By SDS–PAGE combined with mass spectrometry, two interacting proteins were identified: βIII tubulin and myelin basic protein. While myelin basic protein bound to all three P2X subunits, βIII tubulin interacted exclusively with the P2X 2 subunit. The tubulin binding domain could be confined to a proline-rich segment (amino acids 371–412) of the P2X 2 subunit. Our results suggest a role for microtubules in the cellular localisation of the P2X 2 receptor.

Molecular mapping of the determinants involved in human Staufen-ribosome association.

The human double-stranded (ds) RNA-binding protein Staufen (hStau) is considered to have a role in RNA transport and its localization. By using sedimentation analysis on sucrose gradients, we showed that the Staufen isoform with an apparent molecular mass of 55 kDa (Stau(55)) co-fractionated with ribosomes and associated with both the 40 and 60 S ribosomal subunits, suggesting that the Staufen isoform hStau(55) plays some role in translation. To map the determinant(s) involved in this association, we generated a series of deletion mutants and analysed their subcellular distribution by cell fractionation and fluorescent immunomicroscopy. Our results demonstrated that multiple determinants promote hStau(55)-ribosome association via both an RNA-binding-dependent mechanism and protein-protein interaction. The RNA-binding activity of the ds RNA-binding protein domain 3 (dsRBD3) but not that of dsRBD4 is the first determinant. Although necessary for stable association with ribosomes, dsRBD3 alone is not sufficient and needs other determinants as co-factors. Consistently, when expressed together, dsRBD4 and the tubulin-binding domain constitute the minimal Stau(55)/ribosome protein-protein association domain. This region of Stau(55) is sufficient to associate with ribosomes independently, but requires the RNA-binding activity of dsRBD3 for complete association. Thus the results are consistent with a putative role for Stau(55) in the regulation of translation.

Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction.

GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.

Rapid colchicine competition-binding scintillation proximity assay using biotin-labeled tubulin.

We have developed a rapid [3H]colchicine competition-binding scintillation proximity assay (SPA) to evaluate antimitotic compounds that bind to the colchicine-binding site on tubulin. The premise of our assay is that compounds will compete with radiolabeled colchicine for the tubulin-binding domain. Biotin-labeled tubulin is incubated first with unlabeled compound and radiolabeled ligand. Streptavidin-labeled SPA beads are added, and the radiolabel associated with tubulin is directly counted with no separation steps. Under our experimental conditions, the dissociation constant of binding (Kd) for colchicine to tubulin was determined to be 1.4 microM, which was consistent with previously reported values. Assay validation was performed by competitively inhibiting [3H]colchicine binding to tubulin with known microtubule inhibitors and comparing their inhibition constants (Ki). Our SPA bead method is a powerful tool since it overcomes the disadvantage of traditional filtration techniques, as there are no separation steps. It is extremely easy to set up, multiple samples can be assayed and supply and labor costs are reduced because of the minimal volume and test reagents used.

Missense point mutations of tau to segregate with FTDP-17 exhibit site-specific effects on microtubule structure in COS cells: a novel action of R406W mutation.

Missense and splicing point mutations have been found in the tau gene in families with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Of these mutations, we examined four exonic missense point mutations (G272V, P301L, V337M and R406W) in 3-repeat or 4-repeat tau isoform on the transfection experiment. The effects of two mutations (G272V or P301L) on microtubules were subtle whereas those of two other mutations (V337M or R406W) were dramatically significant when these two mutations were constructed into 3-repeat tau but not into 4-repeat tau. The R406W mutation induced an alternation of microtubules to form dotted or fragmented forms retaining colocalization of tau with tubulin whereas the V337M mutation predominantly disrupted microtubule networks and diminished colocalization of tau and tubulin. The effect of the mutations on microtubules were thus site-dependent and isoform-dependent. Tau with R406W mutation was found to be colocalized with tubulin without filamentous structures on confocal views, suggesting that the carboxyl region of tau played a different role from tubulin-binding domain on microtubule assemble. Another abnormal property was identified in tau with R406W mutation that failed to suffer phosphorylation. Thus, diverse effects of tau mutations on microtubules may explain the various clinicopathologies of FTDP-17 and related tauopathies.

Tubulin Binding Sites on γ-Tubulin: Identification and Molecular Characterization

gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.

The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle.

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

Specific Binding of Acidic Phospholipids to Microtubule-associated Protein MAP1B Regulates Its Interaction with Tubulin

Microtubule-associated protein MAP1B, a major neuronal cytoskeletal protein, is expressed highly during the early stage of brain development and is thought to play an important role in brain development. Although it has been shown that MAP1B localizes both in cytosol and particulate fractions, the underlying molecular mechanism in the membrane localization has yet to be elucidated. In the present study, we show that MAP1B purified from young rat brain can bind to acidic phospholipids, such as phosphatidylserine, but not to a neutral phospholipid, phosphatidylcholine. Furthermore, the binding of MAP1B to taxol-stabilized microtubules was inhibited by the addition of phosphatidylserine or phosphatidylinositol. The addition of phosphatidylcholine showed no effect on the binding of MAP1B to the microtubules. A 120-kDa microtubule-binding fragment of MAP1B was also released from microtubules by the addition of acidic phospholipids. Synthetic peptides derived from the C-terminal half of the tubulin-binding domain, but not that corresponding to the N-terminal half, bound to acidic phospholipids specifically. These results suggest that MAP1B binds to biological membranes through its tubulin-binding site, and the binding may play a regulatory role in MAP1B-microtubule interaction.

Identification of microtubule-associated protein tau isoforms in Alzheimer's paired helical filaments.

AD66 proteins derived from sodium dodecylsulfate (SDS) insoluble paired helical filaments (PHF) were isolated from Alzheimer's brain using a purification procedure developed previously in this laboratory, and characterized by immunologic and chemical cleavage methods. AD66 proteins were immunoreactive with antibodies that recognize the amino terminal, tubulin-binding, and carboxy terminal domains of microtubule-associated protein tau indicating the presence of the entire tau sequence in AD66 proteins. These proteins were reactive with antibody 423 that binds to PHF but not human adult tau. Immunologic and chemical cleavage studies indicated that only two of the six tau isoforms were present in these proteins. AD66 proteins were comprised of tau proteins containing only three tubulin binding domains with either a 29 amino acid insert or no amino terminal insert. For comparative purposes, SDS soluble PHF-tau (A68 proteins) was purified from Alzheimer's brains and normal adult tau purified from control brains. Antibody Alz-50 was immunoreactive with PHF-tau or normal tau regardless of alkaline phosphatase treatment while immunoreactivity was only observed with dephosphorylated AD66 proteins. A second phosphorylated epitope on AD66 proteins but not PHF-tau or normal tau proteins was demonstrated with antibody PHF9. These data suggest that AD66 proteins represent a more phosphorylated form of tau than PHF-tau or normal tau proteins. Two-dimensional gel electrophoresis demonstrated that AD66 proteins have higher apparent molecular weights and lower pI values than normal tau, differences possibly due to the greater phosphorylation observed in these proteins.

Mapmodulin: a possible modulator of the interaction of microtubule-associated proteins with microtubules.

We have purified and characterized a 31-kDa protein named mapmodulin that binds to the microtubule-associated proteins (MAPs) MAP2, MAP4, and tau. Mapmodulin binds free MAPs in strong preference to microtubule-associated MAPs, and appears to do so via the MAP's tubulin-binding domain. Mapmodulin inhibits the initial rate of MAP2 binding to microtubules, a property that may allow mapmodulin to displace MAPs from the path of organelles translocating along microtubules. In support of this possibility, mapmodulin stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact CHO cells. To our knowledge, mapmodulin represents the first example of a protein that can bind and potentially regulate multiple MAP proteins.

Molecular Characterization of p62, a Mitotic Apparatus Protein Required for Mitotic Progression

A 62-kDa (p62) mitotic apparatus-associated protein is important for the proper progression of mitosis in sea urchin embryos (Dinsmore, J. H., and Sloboda, R. D. (1989) Cell 53, 769-780). We have isolated and characterized a full-length p62 cDNA of 3374 base pairs which encodes an extremely acidic polypeptide of 411 amino acids having a calculated Mr of 46,388 and a pI of 4.01; p62 is a unique protein with no significant identity to any known proteins. Southern and Northern blot analyses demonstrate that the gene for p62 is present once in the sea urchin genome and the corresponding mRNA is present in unfertilized eggs and in early embryos through and up to the gastrula stage. Sequence analysis suggests certain regions may participate in chromatin association and microtubule binding, an observation that is consistent with previous immunological data (Ye, X., and Sloboda, R. D. (1995) Cell Motil. Cytoskeleton 30, 310-323) as well as data reported herein. Confocal microscopy reveals that during interphase the protein binds to chromatin in the nuclei of sea urchin eggs. In the germinal vesicles of clam oocytes at prophase of meiosis I, p62 binds to the condensed chromosomes. Currently, truncated clones of p62 are being used to identify the tubulin and chromatin binding domains.

Phosphorylation and dephosphorylation in the proline-rich C-terminal domain of microtubule-associated protein 2.

The C-terminal domain of microtubule-associated protein 2 (MAP2) contains a proline-rich region and the tubulin-binding domain. We have generated antibodies to follow the phosphorylation state of the proline-rich domain. One of these antibodies (no. 305) has been raised against a synthetic peptide P (sequence RTPGTPGTPSY) phosphorylated at the threonine residues. This sequence is present in the proline-rich region of MAP2 and is phosphorylated in vitro by at least three different proline-directed protein kinases: p42mpk, p34cdc2, and GSK3 (glycogen-synthase kinase 3) alpha/beta. The MAP2 sites phosphorylated by these kinases are different, although all of them phosphorylate the C-terminal domain of MAP2 as determined by Staphylococcus aureus V8 protease mapping. Nonphosphorylated peptide P can be phosphorylated in vitro by all three kinases studied with similar efficiency. In high-molecular-mass MAP2, this sequence is highly phosphorylated in vivo at the late stages of rat development. This motif can be rapidly dephosphorylated in vitro by protein-phosphatase 1 (PP1) and 2A (PP2A) catalytic subunits but not by PP2B.

Monoclonal antibody PHF-9 recognizes phosphorylated serine 404 of tau protein and labels paired helical filaments.

Paired helical filaments (PHFs) purified from alzheimer's brain consist of hyperphosphorylated microtubule-associated protein tau. In PHF, phosphorylation occurs at ser/thr tau residues. Several of these ser/thr phosphorylation sites lie immediately C-terminal to the tau tubulin binding domain. The C-terminal ser396 to thr413 tau region contains two or more phosphorylated residues and eight possible ser/thr phosphorylation sites. Immunologic studies and mass spectroscopy have identified ser396 as one of the phosphorylation sites but identification of more C-terminal phosphorylated residues has been hampered by the lack of monoclonal antibodies (Mabs) that recognize defined epitopes in this region. We have raised Mabs against PHF purified from Alzheimer's brain. One of these Mabs, PHF-9, showed phosphorylation-dependent binding to purified PHF and recognized a phosphorylated epitope in the C-terminal portion of cyanogen bromide-digested PHF. Epitope mapping studies employing synthetic tau phosphopeptides indicated that PHF-9 labeled a 13-mer tau peptide phosphorylated at ser404 but not the corresponding non-phosphorylated peptide. PHF-9 demonstrated no immunoreactivity with a synthetic peptide phosphorylated at ser396 indicating that the PHF-9 epitope is C-terminal to ser396. In conclusion, the present study describes a Mab, PHF-9, which recognizes phosphorylated ser404 of tau independently of phosphorylated ser396 and indicates that tau ser404 is phosphorylated in PHF.

The phosphatidylinositol-binding site of microtubule-associated protein MAP2

Recent evidence [Surridge and Burns, Biochemistry (1994) 33, 8051-8057] on the interaction of native and recombinant tau, recombinant MAP2c, and native MAP2 with vesicles prepared from phosphatidylinositol (PtdIns) and other phospholipids demonstrate that MAP2 differs from MAP2c and from tau in having a high-affinity PtdIns-binding site. The location of this site within the MAP2-specific insert peptide, coupled with considerations of the nature of the MAP2 tubulin-binding site, suggests that PtdIns-binding induces a conformational change which alters the MAP2 tubulin-binding domain. Furthermore, the restricted cellular distribution of MAP2 implies that the MAP2:PtdIns interaction may play a central role in modulating the dendritic cytoskeleton.

An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif

Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

A τ Fragment Containing a Repetitive Sequence Induces Bundling of Actin Filaments

Much indirect evidence suggests that the interconnections of actin microfilaments with the microtubule system are mediated by microtubule-associated proteins (MAPs). In this study we provide new data to support the interaction of a specific tubulin-binding domain on tau with actin in vitro. In actin polymerization assays, the synthetic peptide VRSKIGSTENLKHQPGGG, corresponding to the first repetitive sequence of tau protein, increased turbidity at 320 nm in a dose-dependent fashion. A salient feature of the tau peptide-induced assembly process is the formation of a large amount of actin filament bundles, as revealed by electron microscopic analysis. An increase in the tau peptide concentration resulted in a proportional increase in the bundling of actin filaments. It is interesting that a gradual decrease of pH within the range 7.6-4.7 resulted in a higher effect of tau peptide in promoting bundles of actin filaments. A similar pH-dependent effect was observed for tau protein-induced bundling. An analysis of the mechanisms that operate in the peptide induction of actin filament bundles suggests the involvement of electrostatic forces, because the neutralization of epsilon-aminolysyl residues by selective carbamoylation resulted in a complete loss of the peptide induction of actin bundles. The data suggest that a tau repetitive sequence (also found in MAP-2 and MAP-4) containing a common tubulin binding motif may constitute a functional domain on tau for the dynamics of the interconnections between actin filaments and microtubules.

High External Potassium Induces an Increase in the Phosphorylation of the Cytoskeletal Protein MAP2 in Rat Hippocampal Slices

Depolarization induced in rat hippocampal slices by a high concentration of extracellular K+ leads to an increase in the phosphorylation of microtubule-associated protein MAP2. The comparison of the major phosphopeptides derived from in situ and in vitro phosphorylated MAP2 suggests the implication of calcium-dependent protein kinases, including calcium/calmodulin-dependent protein kinase type II and protein kinase C, in the up-phosphorylation of MAP2. In particular, a peptide containing the tubulin-binding domain of the MAP2 molecule may be phosphorylated by protein kinase C. As the association of MAP2 with the cytoskeleton may be regulated by phosphorylation, we suggest that changes in the phosphorylation level of MAP2 might be involved in synaptic remodelling in hippocampal neurons.

N-methyl- d-aspartate stimulates the dephosphorylation of the microtubule-associated protein 2 and potentiates excitatory synaptic pathways in the rat hippocampus

We have studied the effect of brief (50–150 s) applications of N-methyl- d-aspartate (10–100 μM) on the phosphorylated state of the microtubule-associated protein 2 in slices of rat hippocampus. Following a similar experimental protocol we also studied the pattern of excitatory postsynaptic potentials intracellularly recorded in CA1 pyramidal cells elicited by stimulation of the Scharfer collateral-commissural pathway. N-Methyl- d-aspartate treatment produced a marked and specific dephosphorylation of the cytoskeletal microtubule-associated protein 2, which was not due to enhanced proteolytic activity. Dephosphorylation of the microtubule-associated protein 2 affects mainly the tubulin-binding domain of the molecule and seems to be a consequence of the activation of the Ca 2+/calmodulin-dependent phosphatase calcineurin, as it is partially inhibited by calmidazolium but not by okadaic acid. A few minutes after N-Methyl- d-aspartate treatment we observed a23 ± 17% increase in the amplitude of the monosynaptic excitatory postsynaptic potential recorded in the cells and the appearance of a large polysynaptic excitatory postsynaptic potential. Both effects lasted for several tens of minutes. The late polysynaptic potential was not observed when the CA3 and CA1 subfields were surgically separated. Our results indicate that the N-Methyl- d-aspartate receptor activation leads to the dephosphorylation of the microtubule-associated protein 2 via a Ca 2+/calmodulin phosphatase, probably calcineurine. This may, in turn, participate in the potentiation of synaptic efficacy.

A tau-like protein interacts with stress fibers and microtubules in human and rodent cultured cell lines

The cytoskeletal integrity of human and rodent cell lines was analyzed using site-directed monoclonal antibodies prepared from hybridomas. Secreting hybridomas were produced by immunizing mice with synthetic peptides from the C-terminal domain of the beta II-tubulin isotype, beta II(422-434), YQQYQDATADEQG, and the first imperfect repeat from brain tau, Tau-I(187-204), VRSKIGSTENLKHQPGGG. Two hybridomas were selected for this work: MTB6.22, an anti-idiotypic monoclonal antibody, which was obtained from a mouse immunized with the beta II-peptide and recognizes specific tubulin-binding domains on MAP-2 and tau; and Tau-I/1, which recognizes the repetitive binding sequences on tau and MAP-2. Immunoblots of cytoskeletal protein preparations from the five different tumor cell lines studied, showed the interaction of the site-directed antibodies MTB6.22 and Tau-I/1 with a group of proteins that co-migrate with brain tau. Immunoreactive tau components were also identified using an anti-tau monoclonal antibody (clone Tau-2), and several polyclonal anti-tau antibodies that interact with tau epitopes, other than those of the tubulin-binding domains. These tau-like proteins bound to a calmodulin-Sepharose affinity column and were eluted using 2 mM EGTA. Interestingly, washing the extracted cytoskeleton pellet with 5 x 10(-3) M Ca2+ for short periods of time selectively released the tau-like protein components, whilst most of the other cytoskeletal proteins remained in the pellet. On the other hand, immunofluorescence microscopy of detergent-extracted cells showed immunostaining of MAP components that appear to be co-localized in a discrete dot-like distribution along the stress fibers, which were revealed using rhodamine-phallacidin. Further support for the specificity of tau interaction with sites on tubulin and actin polymers was obtained with double-immunofluorescence, using the MAP-reactive monoclonal antibody MTB6.22 and a polyclonal antibody to a tubulin peptide containing part of the tau-binding domain on tubulin. Considering the anti-idiotypic nature of the MTB6.22 monoclonal antibody, our studies indicate that, in all the cell lines analyzed, a tau-like protein component is involved in mediating the interaction of both actin and tubulin polymers.

Phosphorylation, calpain proteolysis and tubulin binding of recombinant human tau isoforms

In this study, the phosphorylation, calpain hydrolysis and tubulin binding of three recombinant human tau isoforms were examined. The three isoforms used in these studies were tau with three (T3) or four (T4) tandemly repeated tubulin binding domains located in the carboxy-terminal half of the molecule; and tau with four-tandem repeats and a 58-amino acid insert in the amino terminus (T4L). Both cAMP-dependent protein kinase (cAMP-PK) and Ca 2+ / calmodulin-dependent protein kinase II (CaMKII) readily phosphorylated the three human tau isoforms, although cAMP-PK phosphorylated them to a significantly greater extent than CaMKII. Phosphorylation of T3, T4 and T4L by cAMP-PK or CaMKII resulted in the slowed migration of the protein bands on sodium dodecyl sulfate-polyacrylamide gels and a shift of the isoelectric variants to more acidic positions on two-dimensional non-equilibrium pH gradient electrophoresis gels compared with controls. However, the phosphorylation-induced changes in the electrophoretic migration of the tau isoforms were unique for each kinase. Two-dimensional phosphopeptide maps and sequential phosphorylation experiments indicate that cAMP-PK phosphorylates sites in the human tau isoforms that are phosphorylated by CaMKII, as well as unique sites that are not phosphorylated by CaMKII. T3, T4 and T4L were hydrolyzed similarly by calpain; however, the calpain proteolysis of the recombinant tau isoforms was significantly faster than the proteolysis of human or bovine tau. Phosphorylation of the isoforms by either cAMP-PK or CaMKII did not alter the rate or extent of calpain proteolysis. Phosphorylation of T3, T4 and T4L by cAMP-PK decreased isoform-tubulin binding compared to controls, however only the results with T3 were statistically significant.