SUMOylation of microtubule-cleaving enzyme KATNA1 promotes microtubule severing and neurite outgrowth.

Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.

PTEN functions by recruitment to cytoplasmic vesicles.

PTEN is proposed to function at the plasma membrane, where receptor tyrosine kinases are activated. However, the majority of PTEN is located throughout the cytoplasm. Here, we show that cytoplasmic PTEN is distributed along microtubules, tethered to vesicles via phosphatidylinositol 3-phosphate (PI(3)P), the signature lipid of endosomes. We demonstrate that the non-catalytic C2 domain of PTEN specifically binds PI(3)P through the CBR3 loop. Mutations render this loop incapable of PI(3)P binding and abrogate PTEN-mediated inhibition of PI 3-kinase/AKT signaling. This loss of function is rescued by fusion of the loop mutant PTEN to FYVE, the canonical PI(3)P binding domain, demonstrating the functional importance of targeting PTEN to endosomal membranes. Beyond revealing an upstream activation mechanism of PTEN, our data introduce the concept of PI 3-kinase signal activation on the vast plasma membrane that is contrasted by PTEN-mediated signal termination on the small, discrete surfaces of internalized vesicles.

HDAC3 inhibition disrupts the assembly of meiotic apparatus during porcine oocyte maturation.

Histone deacetylases (HDACs) are involved in a wide array of biological processes. However, the role of HDAC3 in porcine oocytes remains unclear. In the current study, we examine the effects of HDAC3 inhibition on porcine oocyte maturation using RGFP966, a selective HDAC3 inhibitor. We find that suppression of HDAC3 activity prevents not only the expansion of cumulus cells but also the meiotic progression of oocytes. It is interesting to note that HDAC3 displays a spindle-like distribution pattern as the porcine oocytes enter meiosis. In line with this, confocal microscopy reveals the high frequency of spindle defects and chromosomal congression failure in metaphase oocytes exposed to RGFP966. Moreover, HDAC3 inhibition results in the hyperacetylation of α-tubulin during oocyte meiosis. These findings indicate that HDAC3 activity might control the microtubule stability via the deacetylation of tubulin, which is critical for maintaining the proper spindle assembly, accurate chromosome separation, and orderly meiotic progression during porcine oocyte maturation.

Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules.

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states.

The yeast kinesin-5 Cin8 interacts with the microtubule in a noncanonical manner.

Kinesin motors play central roles in establishing and maintaining the mitotic spindle during cell division. Unlike most other kinesins, Cin8, a kinesin-5 motor in Saccharomyces cerevisiae, can move bidirectionally along microtubules, switching directionality according to biochemical conditions, a behavior that remains largely unexplained. To this end, we used biochemical rate and equilibrium constant measurements as well as cryo-electron microscopy methodologies to investigate the microtubule interactions of the Cin8 motor domain. These experiments unexpectedly revealed that, whereas Cin8 ATPase kinetics fell within measured ranges for kinesins (especially kinesin-5 proteins), approximately four motors can bind each αß-tubulin dimer within the microtubule lattice. This result contrasted with those observations on other known kinesins, which can bind only a single "canonical" site per tubulin dimer. Competition assays with human kinesin-5 (Eg5) only partially abrogated this behavior, indicating that Cin8 binds microtubules not only at the canonical site, but also one or more separate ("noncanonical") sites. Moreover, we found that deleting the large, class-specific insert in the microtubule-binding loop 8 reverts Cin8 to one motor per αß-tubulin in the microtubule. The novel microtubule-binding mode of Cin8 identified here provides a potential explanation for Cin8 clustering along microtubules and potentially may contribute to the mechanism for direction reversal.

Differential effects of the dynein-regulatory factor Lissencephaly-1 on processive dynein-dynactin motility.

Cytoplasmic dynein is the primary minus-end-directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.

Location and functional analysis of the Aspergillus nidulans Aurora kinase confirm mitotic functions and suggest non-mitotic roles.

Filamentous fungi have devastating negative impacts as pathogens and agents of food spoilage but also have critical ecological importance and are utilized for industrial applications. The characteristic multinucleate nature of filamentous fungi is facilitated by limiting if, when and where septation, the fungal equivalent of cytokinesis, occurs. In the model filamentous fungus Aspergillus nidulans septation does not occur immediately after mitosis and is an incomplete process resulting in the formation of a septal pore whose permeability is cell cycle regulated. How mitotic regulators, such as the Aurora kinase, contribute to the often unique biology of filamentous fungi is not well understood. The Aurora B kinase has not previously been investigated in any detail during hyphal growth. Here we demonstrate for the first time that Aurora displays cell cycle dependent locations to the region of forming septa, the septal pore and mature septa as well as the mitotic apparatus. To functionally analyze Aurora, we generated a temperature sensitive allele revealing essential mitotic and spindle assembly checkpoint functions consistent with its location to the kinetochore region and spindle midzone. Our analysis also reveals that cellular and kinetochore Aurora levels increase during a mitotic spindle assembly checkpoint arrest and we propose that this could be important for checkpoint inactivation when spindle formation is prevented. We demonstrate that Aurora accumulation at mature septa following mitotic entry does not require mitotic progression but is dependent upon a timing mechanism. Surprisingly we also find that Aurora inactivation leads to cellular swelling and lysis indicating an unexpected function for Aurora in fungal cell growth. Thus in addition to its conserved mitotic functions our data suggest that Aurora has the capacity to be an important regulator of septal biology and cell growth in filamentous fungi.

Increased Aurora B activity causes continuous disruption of kinetochore-microtubule attachments and spindle instability.

Aurora B kinase regulates the proper biorientation of sister chromatids during mitosis. Lack of Aurora B kinase function results in the inability to correct erroneous kinetochore-microtubule attachments and gives rise to aneuploidy. Interestingly, increased Aurora B activity also leads to problems with chromosome segregation, and overexpression of this kinase has been observed in various types of cancer. However, little is known about the mechanisms by which an increase in Aurora B kinase activity can impair mitotic progression and cell viability. Here, using a yeast model, we demonstrate that increased Aurora B activity as a result of the overexpression of the Aurora B and inner centromere protein homologs triggers defects in chromosome segregation by promoting the continuous disruption of chromosome-microtubule attachments even when sister chromatids are correctly bioriented. This disruption leads to a constitutive activation of the spindle-assembly checkpoint, which therefore causes a lack of cytokinesis even though spindle elongation and chromosome segregation take place. Finally, we demonstrate that this increase in Aurora B activity causes premature collapse of the mitotic spindle by promoting instability of the spindle midzone.

Interplay of enzymatic and structural functions of CaMKII in long-term potentiation.

Since the discovery of long-term potentiation (LTP) about a half-century ago, Ca2+ /CaM-dependent protein kinase II (CaMKII) has been one of the most extensively studied components of the molecular machinery that regulate plasticity. This unique dodecameric kinase complex plays pivotal roles in LTP by phosphorylating substrates through elaborate regulatory mechanisms, and is known to be both necessary and sufficient for LTP. In addition to acting as a kinase, CaMKII has been postulated to have structural roles because of its extraordinary abundance and diverse interacting partners. It now is becoming clear that these two functions of CaMKII cooperate closely for the induction of both functional and structural synaptic plasticity of dendritic spines. Because of its extraordinary abundance within neuronal cells, calmodulin kinase CaMKII has been believed to act as a structural protein as well as an enzyme during synaptic plasticity. In this review, we summarized studies in CaMKII field and provide an insight into how enzymatic and structural functions of CaMKII cooperate with each other for long-term potentiation (LTP) in neurons. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Phosphorylation of threonine 3 on histone H3 by haspin kinase is required for meiosis I in mouse oocytes.

Meiosis I (MI), the division that generates haploids, is prone to errors that lead to aneuploidy in females. Haspin is a kinase that phosphorylates histone H3 on threonine 3, thereby recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an AURKB homolog, are enriched in germ cells, yet their significance in regulating MI is not fully understood. Using inhibitors and overexpression approaches, we show a role for haspin during MI in mouse oocytes. Haspin-perturbed oocytes display abnormalities in chromosome morphology and alignment, improper kinetochore-microtubule attachments at metaphase I and aneuploidy at metaphase II. Unlike in mitosis, kinetochore localization remained intact, whereas the distribution of the CPC along chromosomes was absent. The meiotic defects following haspin inhibition were similar to those observed in oocytes where AURKC was inhibited, suggesting that the correction of microtubule attachments during MI requires AURKC along chromosome arms rather than at kinetochores. Our data implicate haspin as a regulator of the CPC and chromosome segregation during MI, while highlighting important differences in how chromosome segregation is regulated between MI and mitosis.

Modulation of cell morphogenesis by tousled-like kinase in the Drosophila follicle cell.

BACKGROUND: Tousled-like kinase (Tlk) is a conserved serine/threonine kinase regulating DNA replication, chromatin assembly, and DNA repair. Previous studies have suggested that Tlk is involved in cell morphogenesis in vitro. In addition, tlk genetically interact with Rho1, which encodes a key regulator of the cytoskeleton. However, whether Tlk plays a physiological role in cell morphogenesis and cytoskeleton rearrangement remains unknown. RESULTS: In tlk mutant follicle cells, area of the apical domain was reduced. The density of microtubules was increased in tlk mutant cells. The density of actin filaments was increased in the apical region and decreased in the basal region. Because area of the apical domain was reduced, we examined the levels of proteins located in the apical region by using immunofluorescence. The fluorescence intensities of two adherens junction proteins Armadillo (Arm) and DE-cadherin (DE-cad), atypical protein kinase C (aPKC), and Notch, were all increased in tlk mutant cells. The basolateral localized Discs large (Dlg) shifted apically in tlk mutant cells. CONCLUSIONS: Increase of protein densities in the apical region might be resulted from disruption of the cytoskeleton and shrinkage of the apical domain. Together, these data suggest a novel role of Tlk in maintaining cell morphology, possibly through modulating the cytoskeleton.

Histone deacetylase 6 inhibits influenza A virus release by downregulating the trafficking of viral components to the plasma membrane via its substrate, acetylated microtubules.

UNLABELLED: Mammalian cells produce many proteins, such as IFITM3, ISG15, MxA, and viperin, that inhibit influenza A virus (IAV) infection. Here, we show that a new class of host protein, histone deacetylase 6 (HDAC6), inhibits IAV infection. We found that HDAC6-overexpressing cells release about 3-fold less IAV progeny, whereas HDAC6-depleted cells release about 6-fold more IAV progeny. The deacetylase activity of HDAC6 played a role in its anti-IAV function as tubacin, a specific small-molecule inhibitor of HDAC6, increased the release of IAV progeny in a dose-dependent manner. Further, as visualized by electron microscopy, tubacin-treated cells showed an increase in IAV budding at the plasma membrane, the site of IAV assembly. Tubacin is a domain-specific inhibitor and binds to one of the two HDAC6 catalytic domains possessing tubulin deacetylase activity. This indicated the potential involvement of acetylated microtubules in the trafficking of viral components to the plasma membrane. Indeed, as quantified by flow cytometry, there was about a 2.0- to 2.5-fold increase and about a 2.0-fold decrease in the amount of viral envelope protein hemagglutinin present on the plasma membrane of tubacin-treated/HDAC6-depleted and HDAC6-overexpressing cells, respectively. In addition, the viral ribonucleoprotein complex was colocalized with acetylated microtubule filaments, and viral nucleoprotein coimmunoprecipitated with acetylated tubulin. Together, our findings indicate that HDAC6 is an anti-IAV host factor and exerts its anti-IAV function by negatively regulating the trafficking of viral components to the host cell plasma membrane via its substrate, acetylated microtubules. IMPORTANCE: Host cells produce many proteins that have the natural ability to restrict influenza virus infection. Here, we discovered that another host protein, histone deacetylase 6 (HDAC6), inhibits influenza virus infection. We demonstrate that HDAC6 exerts its anti-influenza virus function by negatively regulating the trafficking of viral components to the site of influenza virus assembly via its substrate, acetylated microtubules. HDAC6 is a multisubstrate enzyme and regulates multiple cellular pathways, including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of a wide range of diseases. Influenza virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza virus strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza virus potential of endogenous HDAC6 to inhibit influenza virus infection.

Modeling of sensing potency of cytoskeletal systems decorated with metabolic enzymes.

The highly dynamic cytoskeleton interacts with enzymes and other proteins that are involved in metabolic or signaling pathways. These interactions can influence the structural and functional characteristics of the partners at the microscopic level of individual proteins and polymers. In this work the functional consequences of such interactions have been studied at the macroscopic level in order to evaluate the integrative and regulatory roles of the metabolic pathways associated with the microtubule cytoskeleton. Here we present mathematical models of the interactions between a hypothetical metabolic pathway and microtubule assembly, and explore for the first time the functional consequences of these interactions in distinct situations. The models include kinetic constants of the individual steps and testable, relevant parameters which allow the quantification of the coupled processes at the microscopic and macroscopic levels. For example our kinetic model for the self-assembly of microtubules reproduces the alteration of the time-dependent turbidity caused by pyruvate kinase binding. Our data reveal the power of a mechanistic description of a filamentous system to explain how cells sense the state of metabolic and other pathways.

Tubers from patients with tuberous sclerosis complex are characterized by changes in microtubule biology through ROCK2 signalling.

Most patients with tuberous sclerosis complex (TSC) develop cortical tubers that cause severe neurological disabilities. It has been suggested that defects in neuronal differentiation and/or migration underlie the appearance of tubers. However, the precise molecular alterations remain largely unknown. Here, by combining cytological and immunohistochemical analyses of tubers from nine TSC patients (four of them diagnosed with TSC2 germline mutations), we show that alteration of microtubule biology through ROCK2 signalling contributes to TSC neuropathology. All tubers showed a larger number of binucleated neurons than expected relative to control cortex. An excess of normal and altered cytokinetic figures was also commonly observed. Analysis of centrosomal markers suggested increased microtubule nucleation capacity, which was supported by the analysis of an expression dataset from cortical tubers and control cortex, and subsequently linked to under-expression of Rho-associated coiled-coil containing kinase 2 (ROCK2). Thus, augmented microtubule nucleation capacity was observed in mouse embryonic fibroblasts and human fibroblasts deficient in the Tsc2/TSC2 gene product, tuberin. Consistent with ROCK2 under-expression, microtubule acetylation was found to be increased with tuberin deficiency; this alteration was abrogated by rapamycin treatment and mimicked by HDAC6 inhibition. Together, the results of this study support the hypothesis that loss of TSC2 expression can alter microtubule organization and dynamics, which, in turn, deregulate cell division and potentially impair neuronal differentiation.

Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes.

Cellulose synthase (CESA) complexes can be observed by live-cell imaging to move with trajectories that parallel the underlying cortical microtubules. Here we report that CESA interactive protein 1 (CSI1) is a microtubule-associated protein that bridges CESA complexes and cortical microtubules. Simultaneous in vivo imaging of CSI1, CESA complexes, and microtubules demonstrates that the association of CESA complexes and cortical microtubules is dependent on CSI1. CSI1 directly binds to microtubules as demonstrated by in vitro microtubule-binding assay.

Microtubule-severing enzymes at the cutting edge.

ATP-dependent severing of microtubules was first reported in Xenopus laevis egg extracts in 1991. Two years later this observation led to the purification of the first known microtubule-severing enzyme, katanin. Katanin homologs have now been identified throughout the animal kingdom and in plants. Moreover, members of two closely related enzyme subfamilies, spastin and fidgetin, have been found to sever microtubules and might act alongside katanins in some contexts (Roll-Mecak and McNally, 2010; Yu et al., 2008; Zhang et al., 2007). Over the past few years, it has become clear that microtubule-severing enzymes contribute to a wide range of cellular activities including mitosis and meiosis, morphogenesis, cilia biogenesis and disassembly, and migration. Thus, this group of enzymes is revealing itself to be among the most important of the microtubule regulators. This Commentary focuses on our growing understanding of how microtubule-severing enzymes contribute to the organization and dynamics of diverse microtubule arrays, as well as the structural and biophysical characteristics that afford them the unique capacity to catalyze the removal of tubulin from the interior microtubule lattice. Our goal is to provide a broader perspective, focusing on a limited number of particularly informative, representative and/or timely findings.

Arabidopsis thaliana histone deacetylase 14 (HDA14) is an α-tubulin deacetylase that associates with PP2A and enriches in the microtubule fraction with the putative histone acetyltransferase ELP3.

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/ß-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.

ILK: a pseudokinase with a unique function in the integrin-actin linkage.

ILK (integrin-linked kinase) is a central component of cell-matrix adhesions and an important regulator of integrin function. It forms a ternary complex with two other adaptor proteins, PINCH (particularly interesting cysteine- and histidine-rich protein) and parvin, forming the IPP (ILK-PINCH-parvin) complex that regulates the integrin-actin linkage as well as microtubule dynamics. These functions are essential for processes such as cell migration and matrix remodelling. The present review discusses the recent advances on the structural and functional characterization of ILK and the long-standing debate regarding its reported kinase activity.

PAR-1/MARK: a kinase essential for maintaining the dynamic state of microtubules.

The serine/threonine kinase, PAR-1, is an essential component of the evolutionary-conserved polarity-regulating system, PAR-aPKC system, which plays indispensable roles in establishing asymmetric protein distributions and cell polarity in various biological contexts (Suzuki, A. and Ohno, S. (2006). J. Cell Sci., 119: 979-987; Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). PAR-1 is also known as MARK, which phosphorylates classical microtubule-associated proteins (MAPs) and detaches MAPs from microtubules (Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). This MARK activity of PAR-1 suggests its role in microtubule (MT) dynamics, but surprisingly, only few studies have been carried out to address this issue. Here, we summarize our recent study on live imaging analysis of MT dynamics in PAR-1b-depleted cells, which clearly demonstrated the positive role of PAR-1b in maintaining MT dynamics (Hayashi, K., Suzuki, A., Hirai, S., Kurihara, Y., Hoogenraad, C.C., and Ohno, S. (2011). J. Neurosci., 31: 12094-12103). Importantly, our results further revealed the novel physiological function of PAR-1b in maintaining dendritic spine morphology in mature neurons.

Kinesin Kar3Cik1 ATPase pathway for microtubule cross-linking.

Kar3Cik1 is a Saccharomyces cerevisiae kinesin-14 that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet maintains mitotic spindle stability by cross-linking anti-parallel interpolar MTs. Kar3 contains both an ATP- and a MT-binding site, yet there is no evidence of a nucleotide-binding site in Cik1. Presteady-state and steady-state kinetic experiments were pursued to define the regulation of Kar3Cik1 interactions with the MT lattice expected during interpolar MT cross-linking. The results reveal that association of Kar3Cik1 with the MT occurs at 4.9 µM(-1) s(-1), followed by a 5-s(-1) structural transition that limits ADP release from the Kar3 head. Mant-ATP binding occurred at 2.1 µM(-1) s(-1), and the pulse-chase experiments revealed an ATP-promoted isomerization at 69 s(-1). ATP hydrolysis was observed as a rapid step at 26 s(-1) and was required for the Kar3Cik1 motor to detach from MT. The conformational change at 5 s(-1) that occurred after Kar3Cik1 MT association and prior to ADP release was hypothesized to be the rate-limiting step for steady-state ATP turnover. We propose a model in which Kar3Cik1 interacts with the MT lattice through an alternating cycle of Cik1 MT collision followed by Kar3 MT binding with head-head communication between Kar3 and Cik1 modulated by the Kar3 nucleotide state and intramolecular strain.