Activation loop phosphorylation of a protein kinase is a molecular marker of organelle size that dynamically reports flagellar length

Abstract

Specification of organelle size is crucial for cell function, yet we know little about the molecular mechanisms that report and regulate organelle growth and steady-state dimensions. The biflagellated green alga Chlamydomonas requires continuous-length feedback to integrate the multiple events that support flagellar assembly and disassembly and at the same time maintain the sensory and motility functions of the organelle. Although several length mutants have been characterized, the requisite molecular reporter of length has not been identified. Previously, we showed that depletion of Chlamydomonas aurora-like protein kinase CALK inhibited flagellar disassembly and that a gel-shift-associated phosphorylation of CALK marked half-length flagella during flagellar assembly. Here, we show that phosphorylation of CALK on T193, a consensus phosphorylation site on the activation loop required for kinase activity, is distinct from the gel-shift-associated phosphorylation and is triggered when flagellar shortening is induced, thereby implicating CALK protein kinase activity in the shortening arm of length control. Moreover, CALK phosphorylation on T193 is dynamically related to flagellar length. It is reduced in cells with short flagella, elevated in the long flagella mutant, lf4, and dynamically tracks length during both flagellar assembly and flagellar disassembly in WT, but not in lf4. Thus, phosphorylation of CALK in its activation loop is implicated in the disassembly arm of a length feedback mechanism and is a continuous and dynamic molecular marker of flagellar length during both assembly and disassembly.

Keywords: aurora kinase; cilia and flagella; cilia length; flagellar length control; organelle size control.

Fig. 1.

T-loop T193 phosphorylation is required for CALK activity. (A) A diagram of CALK domain structures showing the protein kinase domain and residues critical for CALK activity. (B) T193 is required for CALK activity. Bacterially expressed WT and mutated forms of CALK were assayed for kinase activity in vitro with MBP as substrate. SDS/PAGE analysis with Coomassie blue staining showed equal loading. (C) T193 is phosphorylated in WT CALK but not in kinase-dead and T-loop mutants. Bacterially expressed WT and mutated forms of CALK were analyzed by immunoblotting with anti-pCALK and CALK antibodies. pCALK stains CALK phosphorylated at T193, whereas CALK stains total CALK. (D) Endogenous CALK T193 is phosphorylated at steady state. Steady-state cell lysates were incubated with phosphatase followed by immunoblot analysis with anti-pCALK and CALK antibodies.

Fig. 2.

The CALK gel shift is associated with C-terminal phosphorylation, but not with T193 phosphorylation. (A) The C-terminal domain is required for CALK gel shift-associated phosphorylations. Bacterially expressed and His-tagged WT and mutated forms of CALK were incubated with buffer or with a lysate from deflagellated Chlamydomonas cells in the presence of ATP. After incubation for 15 min at 30 °C, samples were analyzed by immunoblot analysis with anti-His antibody. Buf, buffer; Lys, cell lysate. (B) CALK C-terminal region is required for the gel-shift–associated phosphorylation that occurs upon flagellar detachment. Chlamydomonas cells expressing HA-tagged WT CALK or C-terminally truncated CALK (CALK1-341-HA) were deflagellated and subjected to immunoblot analysis with anti-HA and anti-CALK antibodies. Endogenous CALK is shown as a control. Left arrows indicate CALK-HA and right arrow, CALK1-341-HA. (C) The gel shift-associated phosphorylation during flagellar shortening occurs on the C-terminal region of CALK. Chlamydomonas cells expressing HA-tagged, C-terminally truncated CALK were exposed to 20 mM NaPPi for 5 min to trigger activation of the flagellar shortening pathway and analyzed by immunoblotting. (D) T193 phosphorylation does not cause a CALK gel shift. Chlamydomonas cells expressing HA-tagged WT CALK or kinase-dead mutant K66R-HA CALK were treated with NaPPi for 5 min, followed by immunoprecipitation with HA antibody and immunoblot analysis.

Fig. 3.

Activation of the flagellar shortening pathway induces a rapid increase in T193 phosphorylation, and shortening is accompanied by a concomitant decrease in levels of CALK phosphorylated on T193. (A) The level of CALK phosphorylated on T193 increases when flagellar shortening is triggered by NaPPi and during zygotic development. Samples harvested at the indicated times after NaPPi treatment or after mixing gametes of opposite mating types were analyzed by immunoblotting. (B) Dynamic changes of CALK T193 phosphorylation during flagellar shortening induced by NaPPi. Cells were induced to shorten their flagella by adding 20 mM NaPPi. At the times indicated during flagellar shortening, samples were analyzed for flagellar length (Upper) and for CALK phosphorylation by immunoblotting (Lower). Flagellar length data shown here and below are represented as mean ± SD). (C) The relative amount of CALK phosphorylated on T193 is proportional to flagellar length during flagellar shortening. The relative amounts of pCALK as determined by densitometry from immunoblotting with pCALK and CALK antibodies from two experiments are plotted against flagellar length during shortening. (D) CALK T193 phosphorylation in cells with short or absent flagella. Aflagellate mutants bld1 and bld2, aflagellate cells grown on agar plates, aflagellate cells generated by pH shock, and the short flagella mutant shf2 were analyzed by immunoblotting with anti-CALK and anti-pCALK antibodies. deflag, deflagellation by pH shock; pdf, predeflagellation. (E) Localization of CALK T193 phosphorylated form at the basal body region. Control cells and cells deflagellated by pH shock were immunostained with pCALK and anti–α-tubulin antibodies. (Insets) Higher magnification views of basal body regions.

Fig. 4.

Length-dependent regulation of phosphorylation state of CALK T193. (A) Change in levels of CALK phosphorylated on T193 during flagellar elongation. At the indicated times during flagellar regeneration after pH shock, samples were analyzed for T193 phosphorylation state by immunoblotting (Top) and for flagellar length (Middle). The ratio of CALK phosphorylated on T193 (pCALK) versus total CALK determined by densitometry is plotted against time (Middle) and flagellar length (Bottom). Data shown are from three independent experiments. (B) Levels of T193 phosphorylated CALK in cells whose flagellar length was fixed by addition of colchicine during flagellar regeneration. Colchicine was added at the indicated times after deflagellation; 2 h later, samples were analyzed for flagellar lengths (Top) and by immunoblot analysis (Middle). The relative amounts of T193 phosphorylated CALK from two independent experiments were quantified by densitometry (Bottom). (C) Analysis of T193 phosphorylation at steady state and during flagellar elongation in the long flagellar mutant lf4-3. lf4-3 cells were deflagellated by mechanical shearing and allowed to regenerate flagella. Cell samples pdf and at the indicated times after deflagellation were analyzed by immunoblotting (Upper) and for flagellar length (Lower). An immunoblot of a WT control sample is also shown. (D) Analysis of T193 phosphorylation during flagellar shortening in lf4-3. Flagellar lengths and pCALK and CALK immunoblot analysis of lf4-3 cells during NaPPi-induced shortening.