Tyrosines in the kinesin-5 head domain are necessary for phosphorylation by Wee1 and for mitotic spindle integrity

Abstract

Mitotic spindle assembly and maintenance relies on kinesin-5 motors that act as bipolar homotetramers to crosslink microtubules. Kinesin-5 motors have been the subject of extensive structure-function analysis, but the regulation of their activity in the context of mitotic progression remains less well understood. We report here that Drosophila kinesin-5 (KLP61F) is regulated by Drosophila Wee1 (dWee1). Wee1 tyrosine kinases are known to regulate mitotic entry via inhibitory phosphorylation of Cdk1. Recently, we showed that dWee1 also plays a role in mitotic spindle positioning through gamma-tubulin and spindle fidelity through an unknown mechanism. Here, we investigated whether a KLP61F-dWee1 interaction could explain the latter role of dWee1. We found that dWee1 phosphorylates KLP61F in vitro on three tyrosines within the head domain, the catalytic region that mediates movement along microtubules. In vivo, KLP61F with tyrosine-->phenylalanine mutations fails to complement a klp61f mutant and dominantly induces spindle defects similar to ones seen in dwee1 mutants. We propose that phosphorylation of the KLP61F catalytic domain by dWee1 is important for the motor's function. This study identifies a second substrate for a Wee1 kinase and provides evidence for phosphoregulation of a kinesin in the head domain.

Figure 1. Endogenous KLP61F and dWee1 interact

Extracts from syncytial wild type embryos were immunoprecipitated with anti-KLP61F serum or mock-precipitated (no Ab IP) and western blotted for KLP61F (A) or dWee1 (B). Samples equivalent to 0.6% and 1.2% of starting extracts were loaded for comparison.

Figure 2. GST-dWee1 phosphorylates KLP61F on tyrosines in the head domain in vitro

(A) Schematic representation of KLP61F with head domain (red), coiled-coil (blue) and tail domain that contains the BimC box (yellow). His-tagged polypeptides used in kinase assays are depicted. (B) His-KLP61F was incubated with varying amounts of GST-dWee1 in kinase reactions in vitro and analyzed for ³²P incorporation. (C) Reaction with equimolar GST-dWee1 and His-KLP61F was western blotted with anti-phosphotyrosine antibody after in vitro kinase assays. (D–E) GST-dWee1 and His-KLP61F polypeptides were incubated in in vitro kinase reactions and analyzed for ³²P incorporation (left panels) and subsequently by western blots for phosphotyrosines (right panels). (F) KLP61F head domain with Y23, Y152 and Y207 mutated to phenylalanines, His-Head^3YF, was incubated with GST-dWee1 in in vitro kinase reactions and analyzed for ³²P incorporation. none = no kinesin; GST-KHC = conventional Kinesin Heavy Chain head domain; His-Head^WT = wild type head domain. (G–H) Western blots for GST (G) or His tags (H) show equivalent amounts of substrates in kinase reactions in (F).

Figure 3. Phospho-accepting tyrosines in the head domain are important in vivo

(A–B) Pupal lethality of klp61f³ mutants (A) and level of aneuploidy in klp61f³ mutant neuroblasts (B) are rescued by a Myc-KLP61F^WT transgene ([WT]; klp61f³) but not a Myc-KLP61F^3YF transgene ([3YF]; klp61f³). Data is represented as percentage of dead pupae (mean +/− SD from 3 embryo collections) in (A) and as percentage of aneuploid nuclei (mean +/− SD of nuclei from 3 larval brains) in (B). * indicates p < 0.01.

Figure 4. Mitotic spindle defects in embryos containing KLP61F^WT or KLP61F^3YF

Embryos from mothers that are wild type, klp61f³ heterozygotes (klp61f), klp61f³ heterozygotes with Myc-KLP61F^WT ([WT]) and klp61f³ heterozygotes with Myc-KLP61F^3YF ([3YF]) were fixed and stained for DNA (blue), α-tubulin (green), and centrosomin (red). Representative images show a normal spindle (A) and defects significantly different among the genotypes: a monopolar spindle (B), an anastral spindle (C), and spindles with multiple MTOCs (F–G) from interior divisions, and a promiscuous microtubule interaction during anaphase in cortical divisions (H). Scale bar = 5 µm. Defects in (A–C) and (F–H) are quantified in (D) and (E) respectively. See Table 1 for numbers. * p < 0.01, ** p < 0.001, and ^ p < 0.05. (I) A model for regulation of KLP61F to maintain mitotic spindle integrity and prevent promiscuous MT interactions. dWee1 may regulate KLP61F activity to bundle parallel (not shown) and/or anti-parallel microtubules to create a more robust spindle (left). Without dWee1 regulation, KLP61F activity is reduced on the spindle, leading to an unstable spindle with microtubule spurs. These microtubule spurs can then interact with neighboring spindles in a syncytium (spur-spindle interaction is not depicted). Reduced KLP61F activity on the spindle is depicted as reduced protein levels on the spindle, but it remains possible that similar level of protein associate with the spindle but display reduced activity.