Kristina Blake-Hodek

Cornell University, Итак, New York, United States

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Publications (6)34.93 Total impact

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    ABSTRACT: eLife digest The most dramatic stage of the cell division cycle is M phase, when the cell splits into two genetically identical daughter cells. If this process goes wrong, the cell might die, so cells employ a complicated regulatory process to ensure that M phase begins and ends at the right time. As with many biological processes, regulation of the cell cycle depends on the activation and inhibition of a range of enzymes. Enzymes act as biological catalysts, binding target molecules (substrates) to active sites so that chemical reactions can take place. However, the activity of the enzyme can be shut down if a different type of molecule, called a competitive inhibitor, binds to the active site. For M phase to proceed, an enzyme called M phase promoting factor adds phosphate groups to hundreds of target proteins. At the end of M phase, a different enzyme, called PP2A-B55, removes these phosphate groups. Cells can enter M phase because an inhibitor called Endosulfine blocks the active site of the PP2A-B55 enzyme. However, the cells need to unblock the PP2A-B55 enzyme at the end of M phase. Williams et al. have now established the mechanism behind this unblocking of the PP2A-B55 enzyme. One basis for this mechanism is that Endosulfine works as an inhibitor only when it is phosphorylated (contains a phosphate group). Throughout M phase, a plentiful supply of newly phosphorylated Endosulfine inhibitor molecules binds very tightly to the active sites of the PP2A-B55 enzyme molecules, blocking the enzyme’s more loosely binding substrates from accessing the active sites. The second basis for this mechanism is that PP2A-B55 can also slowly remove the phosphate groups from Endosulfine molecules bound at the active site. In other words, phosphorylated Endosulfine works as an inhibitor only because it is really a substrate with special properties. It binds very tightly to the active site, where it is destroyed very slowly. For this reason, Williams et al. have named the process inhibition by unfair competition. In the final stages of M phase, the cell cannot produce any more phosphorylated Endosulfine molecules, and the PP2A-B55 enzyme can then destroy all the existing inhibitors. Even though this reaction is relatively slow, it is still achieved within a couple of minutes. After its active site is no longer blocked, the PP2A-B55 enzyme is then free to remove the phosphate groups from the target proteins, and M phase can come to an end. DOI: http://dx.doi.org/10.7554/eLife.01695.002
    eLife Sciences 03/2014; 3:e01695. DOI:10.7554/eLife.01695 · 8.52 Impact Factor
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    ABSTRACT: In vertebrates, mitotic and meiotic M phase is facilitated by the kinase Greatwall (Gwl), which phosphorylates a conserved sequence in the effector Endosulfine (Endos). Phosphorylated Endos inactivates the phosphatase PP2A/B55 to stabilize M-phase-specific phosphorylations added to many proteins by cyclin-dependent kinases (CDKs). We show here that this module functions essentially identically in Drosophila melanogaster and is necessary for proper mitotic and meiotic cell division in a wide variety of tissues. Despite the importance and evolutionary conservation of this pathway between insects and vertebrates, it can be bypassed in at least two situations. First, heterozygosity for loss-of-function mutations of twins, which encodes the Drosophila B55 protein, suppresses the effects of endos or gwl mutations. Several types of cell division occur normally in twins heterozygotes in the complete absence of Endos or the near absence of Gwl. Second, this module is nonessential in the nematode Caenorhaditis elegans. The worm genome does not contain an obvious ortholog of gwl, although it encodes a single Endos protein with a surprisingly well-conserved Gwl target site. Deletion of this site from worm Endos has no obvious effects on cell divisions involved in viability or reproduction under normal laboratory conditions. In contrast to these situations, removal of one copy of twins does not completely bypass the requirement for endos or gwl for Drosophila female fertility, although reducing twins dosage reverses the meiotic maturation defects of hypomorphic gwl mutants. These results have interesting implications for the function and evolution of the mechanisms modulating removal of CDK-directed phosphorylations.
    Genetics 05/2012; 191(4):1181-97. DOI:10.1534/genetics.112.140574 · 4.87 Impact Factor
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    ABSTRACT: The atypical AGC kinase Greatwall (Gwl) mediates a pathway that prevents the precocious removal of phosphorylations added to target proteins by M phase-promoting factor (MPF); Gwl is thus essential for M phase entry and maintenance. Gwl itself is activated by M phase-specific phosphorylations that are investigated here. Many phosphorylations are nonessential, being located within a long nonconserved region, any part of which can be deleted without effect. Using mass spectrometry and mutagenesis, we have identified 3 phosphorylation sites (phosphosites) critical to Gwl activation (pT193, pT206, and pS883 in Xenopus laevis) located in evolutionarily conserved domains that differentiate Gwl from related kinases. We propose a model in which the initiating event for Gwl activation is phosphorylation by MPF of the proline-directed sites T193 and T206 in the presumptive activation loop. After this priming step, Gwl can intramolecularly phosphorylate its C-terminal tail at pS883; this site probably plays a role similar to that of the tail/Z motif of other AGC kinases. These events largely (but not completely) explain the full activation of Gwl at M phase.
    Molecular and Cellular Biology 02/2012; 32(8):1337-53. DOI:10.1128/MCB.06525-11 · 5.04 Impact Factor
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    ABSTRACT: Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.
    Molecular biology of the cell 05/2011; 22(13):2157-64. DOI:10.1091/mbc.E11-01-0008 · 5.98 Impact Factor
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    Kristina A Blake-Hodek, Lynne Cassimeris, Tim C Huffaker
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    ABSTRACT: Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.
    Molecular biology of the cell 06/2010; 21(12):2013-23. DOI:10.1091/mbc.E10-02-0083 · 5.98 Impact Factor
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    ABSTRACT: Microtubule plus-end tracking proteins (+TIPs) are a diverse group of molecules that regulate microtubule dynamics and interactions of microtubules with other cellular structures. Many +TIPs have affinity for each other but the functional significance of these associations is unclear. Here we investigate the physical and functional interactions among three +TIPs in S. cerevisiae, Stu2, Bik1, and Bim1. Two-hybrid, coimmunoprecipitation, and in vitro binding assays demonstrate that they associate in all pairwise combinations, although the interaction between Stu2 and Bim1 may be indirect. Three-hybrid assays indicate that these proteins compete for binding to each other. Thus, Stu2, Bik1, and Bim1 interact physically but do not appear to be arranged in a single unique complex. We examined the functional interactions among pairs of proteins by comparing cytoplasmic and spindle microtubule dynamics in cells lacking either one or both proteins. On cytoplasmic microtubules, Stu2 and Bim1 act cooperatively to regulate dynamics in G1 but not in preanaphase, whereas Bik1 acts independently from Stu2 and Bim1. On kinetochore microtubules, Bik1 and Bim1 are redundant for regulating dynamics, whereas Stu2 acts independently from Bik1 and Bim1. These results indicate that interactions among +TIPS can play important roles in the regulation of microtubule dynamics.
    Molecular Biology of the Cell 07/2006; 17(6):2789-98. DOI:10.1091/mbc.E05-09-0892 · 4.55 Impact Factor