1. Volume 143, Issue 6, Pages 966-977 (December 2010)
Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids 
Katarina Moravcevic, Jeannine M. Mendrola, Karl R. Schmitz, Yu-Hsiu Wang, David Slochower, Paul A. Janmey, Mark A. Lemmon 
Cell 
Volume 143, Issue 6, Pages (December 2010)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
A C-Terminal Domain in Kcc4p Binds Phospholipids and Associates with Cell Membranes
(A) A C-terminal 160 aa Kcc4p fragment (residues 877–1037) is necessary and sufficient for membrane recruitment of Ha-RasQ61L fusions, rescuing 37°C growth of cdc25ts yeast cells. Serial dilutions of yeast cultures expressing each Kcc4p fragment were spotted in duplicate onto selection plates and incubated at 25°C or 37°C.
(B) The same C-terminal Kcc4p fragment, fused to GFP, shows plasma membrane localization in S. cerevisiae and HeLa cells.
(C) SPR studies of Kcc4p901–1037 binding to DOPC membranes containing 10% (mole/mole) PtdIns(4,5)P2 (KD = 10.6 ± 1.1 μM), 20% (mole/mole) phosphatidic acid (KD = 10.2 ± 0.3 μM), or 20% (mole/mole) PtdSer (KD = 7.8 ± 3.4 μM). Binding curves are representative of at least three independent experiments, and mean KD values ± standard deviation are quoted (Table S2).
(D) SPR signals at saturation show that maximal Kcc4p901–1037 binding scales with the negative charge density in immobilized membranes. Mean Bmax values ± standard deviations (for >3 experiments) are plotted for membranes containing the noted percentages (mole/mole) of PtdIns(4,5)P2 (valence −4 at pH 7.4) and PtdSer (valence −1 at pH 7.4).
(E) In vesicle sedimentation studies, His6-Kcc4p901–1037 (at 50 μM) binds small unilamellar vesicles containing 20% (mole/mole) phosphatidylinositol (PtdIns) or 20% (mole/mole) PtdSer in a brominated PC background, but not to phosphatidylethanolamine (PE). At 500 μM “total available lipid,” 100 μM of PtdIns, PE, or PtdSer is available for binding on the vesicle outer leaflet. Mean ± standard deviation is plotted for at least three independent experiments.
Figure S1 and Tables S1A and S1B summarize results for other potential phosphoinositide-binding proteins.
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4. Figure 2
The Membrane-Targeting Domain of Kcc4p Is Conserved in Gin4p and Hsl1p
(A) Alignment of C-terminal fragments from the three S. cerevisiae septin-associated kinases Kcc4p, Gin4p, and Hsl1p. Acidic residues are red, basic blue, hydrophobic green, and hydrophilic plum. Colored blocks or text denote positions at which two or more residues are identical or similar, respectively. See also Figure S2.
(B) Ras Rescue studies of Gin4p943–1142 and Hsl1p1358–1518.
(C) GFP/Gin4p1003–1142 and GFP/Hsl1p1358–1518 localize to the plasma membrane in S. cerevisiae cells.
(D) SPR studies show that GST/Gin4p943–1142 binds DOPC membranes containing 20% (mole/mole) phosphatidic acid (KD = 5.7 ± 0.5 μM), 20% PtdSer (KD = 8.6 ± 2.6 μM), or 10% PtdIns(4,5)P2 (KD = 4.7 ± 0.3 μM). Binding curves are representative of at least three independent experiments. Note that GST dimerization causes overestimation of apparent binding affinity in this assay (Yu et al., 2004).
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5. Figure 3
Phosphatidylserine Depletion Reduces Membrane Association of Kcc4p877–1037, Gin4p1003–1142, and Hsl1p1358–1518
Localization of GFP-fused Kcc4p877–1037, Gin4p1003–1142, and Hsl1p1358–1518 in wild-type yeast cells (left) and in cho1Δ cells, which lack PtdSer. The lactadherin C2 domain was used as a control probe for PtdSer (Yeung et al., 2008). The five panels shown for each GFP fusion in cho1Δ cells reflect the range of localization phenotypes observed, illustrating reduced plasma membrane association. Figure S3 shows that reducing phosphoinositide levels has no such effect.
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6. Figure 4 The Kcc4p C Terminus Adopts a KA1 Domain Fold
(A) Cartoon representation of Kcc4p917–1037 structure. Helices αN, α1, and α2 are marked, as are strands β1–β5. Two orthogonal views are shown. See also Figure S4.
(B) NMR structure (Tochio et al., 2006) of the KA1 domain from mouse MARK3 (PDB ID 1UL7), in the same orientations used in (A) for Kcc4p917–1037.
(C) Cα overlay of MARK3-KA1 (cyan) with Kcc4p917–1037 (magenta). The N-terminal part of Kcc4p917–1037, including helix αN, was removed for clarity.
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7. Figure 5 KA1 Domains from Human MARK/PAR1 Kinases Bind Phospholipids
(A) KA1 domains from human MARK1 (aa 648–795), human MARK3 (aa 589–729), and human MELK (aa 500–651) all drive membrane recruitment of Ha-RasQ61L fusions in Ras rescue studies.
(B) GFP-fused human MARK1 and MARK3 KA1 domains show plasma membrane localization in HeLa cells. Unexplained nuclear localization of the MELK-KA1 fusion made interpretation of its behavior difficult (not shown).
(C) GFP-fused KA1 domains from human MARK1, MARK3, and MELK show robust plasma membrane localization in S. cerevisiae cells, which is diminished in cho1Δ cells that lack PtdSer. Mean FPM/FCyt ratios for each experiment (±standard deviation) are quoted in individual panels. Figure S5 shows that manipulating phosphoinositide levels in yeast cells does not affect membrane targeting of MARK family KA1 domains.
(D) Purified MARK1-KA1 binds membranes containing phosphatidic acid (20%), PtdSer (20%), or PtdIns(4,5)P2 (10%) in SPR studies. Binding curves are representative of at least three independent experiments. Mean apparent KD values (±standard deviation) are listed in Table S2.
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8. Figure 6
Potential Phospholipid-Binding Sites on Kcc4p and MARK1 KA1 Domains
(A) Kcc4p-KA1 is shown in surface representation (left: with electrostatic surface potential—blue, positive; red, negative) and in cartoon form (right: same orientation). The two ordered sulfate ions (SO4#1 and SO4#2) and the glycerol molecule close to SO4#1 are marked, as is the β3/β4 loop. Figure S4 shows the tartrate ion that replaces SO4#1 and the glycerol in another crystal form. Noted residues were mutated in pairs to serine, expression confirmed by western blotting (not shown), and effects on plasma membrane localization of GFP fusions assessed in wild-type yeast cells (right). Double mutations marked with red asterisks showed significantly reduced FPM/FCyt ratios compared with wild-type Kcc4p-KA1 (mean FPM/FCyt = 1.7 ± 0.3). FPM/FCyt values for mutated variants were 0.81 ± 0.09 (K930S/K932S), 0.74 ± 0.03 (K959S/K964S), 0.80 ± 0.15 (K964S/K978S), 0.92 ± 0.14 (K1007S/K1010S), 0.96 ± 0.06 (K1016S/K1020S). Residues implicated in membrane binding are colored black, whereas those at which mutations did not influence targeting are gray.
(B) Crystal structure of human MARK1-KA1 (Table S3), shown in the same orientation as in (A). Compared with an FPM/FCyt ratio of 2.0 ± 0.4 for wild-type MARK1-KA1, mutated variants denoted by red asterisks gave FPM/FCyt values of 0.90 ± 0.20 (R698S/R701S), 0.93 ± 0.19 (R771A/K773A), and 0.99 ± 0.04 (K773S/R774S). Figure S6 describes effects of these mutations on in vitro binding.
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9. Figure 7 Role of the KA1 Domain in Kcc4p and Gin4p
(A) Localization of wild-type and KA1 domain-mutated intact GFP/Kcc4p in wild-type yeast cells (normal) and PtdSer-deficient cho1Δ cells. Additional images and western blot confirmation of intact protein expression are shown in Figure S7.
(B) Yeast cells lacking Gin4p (gin4Δ) show an elongated bud phenotype (left). Overexpressed GFP-fused full-length Gin4p in gin4Δ cells rescues this aberrant elongated-bud morphology and is found at the bud neck in all cells. By contrast, GFP/Gin4pΔKA1 fails to rescue the gin4Δ phenotype and remains diffuse in the cytoplasm. Examining at least 200 cells in several experiments, the elongated phenotype was seen in 69% of gin4Δ cells expressing GFP alone, 78% expressing GFP/Gin4pΔKA1, and just 39% of those expressing GFP/Gin4p.
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10. Figure S1
Identification of Phospholipid-Binding Domains in S. cerevisiae Proteins, Related to Figure 1
(A) Ras Rescue data for the 33 S. cerevisiae proteins (of 62 tested) capable of recruiting Ha-RasQ61L to yeast cell membranes (see also Table S1A). Serial dilutions of cdc25ts yeast cultures expressing the noted protein fused to non-farnesylated Ha-RasQ61L were spotted onto duplicate selection plates lacking leucine and incubated at the permissive (25°C: left) and restrictive (37°C: right) temperature for 4–5 days. Controls are shown at the top. The phospholipase C-δ1 PH domain (PLCδ-PH), binds PtdIns(4,5)P2 with high affinity and serves as a positive control (Yu et al., 2004). The dynamin PH domain binds phospholipids very weakly, and serves as a negative control (Isakoff et al., 1998; Yu et al., 2004).
All 33 yeast proteins listed in this figure gave positive Ras rescue results. Those within the yellow box allowed subsequent identification of subregions responsible for phospholipid binding (see B). Those in the cyan box bound phospholipids in overlay experiments, and subdomains responsible could be identified in a few cases (see B). Those listed in the green box failed to bind phospholipids in overlay or other experiments. Those in the gray box could not be expressed in sufficiently large quantities for in vitro analysis of phospholipid binding.
(B) In addition to the Kcc4p KA1 domain (Figure 1), we identified subregions for Cam1p, Dps1p, Stp22p, and Rgd1p that appear necessary and sufficient for membrane targeting. For Cam1p, this is a GST domain. For Stp22p, the N-terminal 190 amino acids, which constitute an UBC-like domain, appear sufficient for membrane targeting. For Rgd1p, the amino-terminal 324 amino acids (an F-BAR domain) are necessary and sufficient for membrane targeting.
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11. Figure S2
Structure-Based Sequence Alignment of Human and Yeast KA1 Domains, Related to Figure 2
Mammalian and yeast KA1 domains are aligned, with sequence numbers provided and secondary structure elements listed above the sequence. The structure-based sequence alignment was generated using ESPript (Gouet et al., 1999). The alignment of MARK/PAR1/Kin kinase KA1 domains closely resembles that reported following determination of the mMARK3-KA1 structure (Tochio et al., 2006). Alignment of the Kcc4p, Gin4p, and Hsl1p KA1 domains is based on structural studies described here. Red residues within vertical boxes represent positions at which amino acid type is most well conserved across the alignment. Secondary structure elements αN (seen only in Kcc4p-KA1), α1, α2 and β1-β5 are marked. TT denotes a β turn. Sequence similarity is greatest toward the C terminus, especially in the β5/α2 region, which constitutes a major binding site for anions (Figure S4) at which we argue phospholipids are likely to bind. Residues in Kcc4p-KA1 that contribute to the binding site for SO4#1, tartrate, or glycerol, and contribute to membrane association (see Figures 6 and Figure S4) are boxed in green. Those that contribute to the binding site for SO4#2 (not conserved in hMARK1-KA1) are boxed in orange. Residues in hMARK1-KA1 that form part of the basic patch and contribute to membrane association (Figure 6) are also colored green. These lie in areas similar to the Kcc4p-KA1 SO4#1 binding site. Note the conserved positive charge in β5 and the relatively conserved S/T residues in α5 that contact glycerol in Kcc4p-KA1.
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12. Figure S3
Membrane Targeting of Kcc4p877–1037, Gin4p1003–1142, and Hsl1p1358–1518 in Yeast Strains with Altered Phosphoinositide Levels, Related to Figure 3
Localization of GFP-fused Kcc4p877–1037, Gin4p1003–1142, and Hsl1p1358–1518 was examined in yeast strains with temperature sensitive mutations in PtdIns 4-kinases (stt4ts and pik1ts) or the major PtdIns4P 5-kinase (mss4ts). Cells were grown to mid-log phase and then incubated for 40 min at either 26°C or 37°C. The Num1p and Osh1p PH domains were used as control probes for PtdIns(4,5)P2 and Golgi PtdIns4P respectively (Yu et al., 2004). All images are representative of at least 75% of expressing cells observed.
PtdIns(4,5)P2 levels are reduced in mss4ts cells by approximately 40% at 26°C, and by more than 80% above the restrictive temperature (Stefan et al., 2002), because of a mutation in the gene encoding the major PtdIns4P 5-kinase (Desrivieres et al., 1998; Homma et al., 1998). Despite these reductions in PtdIns(4,5)P2 levels, plasma membrane localization of the Kcc4p, Gin4p or Hsl1p C-termini is retained with no significant alteration in FPM/FCyt ratios above the restrictive temperature, whereas the PtdIns(4,5)P2-binding Num1p PH domain becomes delocalized (Yu et al., 2004). Similarly, temperature-sensitive mutations in the genes encoding the PtdIns 4-kinases that generate PtdIns4P at the plasma membrane (Stt4p) and Golgi (Pik1p) respectively (Audhya et al., 2000) did not result in altered FPM/FCyt ratios for the Kcc4p, Gin4p or Hsl1p C-termini above the restrictive temperature, whereas control phosphoinositide-dependent membrane probes (Num1p and Osh1p PH domains) became delocalized. Thus, phosphoinositides do not appear to play a dominant role in membrane localization of the C-terminal domains from yeast septin-associated kinases, despite the fact that our focus on Kcc4p came from its initial identification as a phosphoinositide-binding protein (Zhu et al., 2001).
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13. Figure S4
Details of a Potential Phospholipid-Binding Site in the Kcc4p KA1 Domain, Related to Figure 4
The structure of the Kcc4p KA1 domain was obtained from two different crystal forms, grown with sulfate and tartrate as the most prevalent anions (in space groups P21 and P1 respectively: see Table S3). Bound anions were seen in both structures, and were located in a common binding site between β5 and α1 that is therefore implicated as a phospholipid-binding site (see Figure 6).
(A) The Kcc4p-KA1 structure shown in Figure 4 and Figure 6 has two bound sulfate ions (SO4#1 and SO4#2) and glycerol molecules in the model. One sulfate (SO4#1) lies close to the linker between strand β5 and helix α2, and is adjacent to a bound glycerol. This region of the KA1 domain is among the most conserved, and was previously suggested on that basis to represent a binding site in hMARK3-KA1 (Tochio et al., 2006). Side chains of basic residues (K932, K1010, and K1016) interact with the bound sulfate, and the S1014 and T1015 side chains interact with the bound glycerol.
(B) When crystallized in the presence of tartrate, a tartrate ion is seen at the same location, making the same interactions as the glycerol molecule (with S1014 and T1015), plus some of those made by SO4#1 in (A). In addition, the tartrate ion interacts with R988 from the β3/β4 loop. The binding mode of tartrate (or sulfate plus glycerol) suggests that this site represents a phospholipid-binding site, as also indicated by mutational studies presented in Figure 6.
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14. Figure S5
Localization of Human MARK/MELK KA1 Domains in Yeast Cells with Altered Phosphoinositide Levels, Related to Figure 5
Localization of the human MARK1, MARK3, and MELK KA1 domains (fused to GFP) was analyzed in mss4ts cells, stt4ts cells, and stt4ts/pik1ts cells, grown to mid-log phase, and then incubated for 40 min at either 26°C or 37°C before being examined by fluorescence microscopy. Images are representative of >90% of cells observed (from three experiments in each of which > 100 cells were analyzed). As described in Figure S3, levels of PtdIns(4,5)P2 are greatly reduced in mss4ts and stt4ts cells at the restrictive temperature. Plasma membrane and total PtdIns4P levels are greatly reduced at the restrictive temperature in stt4ts and stt4ts/pik1ts cells respectively. In no case did elevating the temperature above the restrictive temperature lead to a significant alteration of FPM/FCyt ratios for the GFP/KA1, by contrast with the impaired plasma membrane localization seen in cho1Δ cells in Figure 5.
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15. Figure S6
Monolayer Penetration of Kcc4p-KA1 and In Vitro Phospholipid Binding of hMARK1-KA1, Related to Figure 6
(A) Monolayer studies were performed using a μTroughS Langmuir trough (Kibron Inc., Helsinki, Finland) essentially as described (Cunningham et al., 2001; Medina et al., 2001). Briefly, 1 ml of buffer (25 mM HEPES, pH 7.5, containing 150 mM NaCl) was placed as subphase solution in a well, and the desired lipid mixtures were spread at the air-buffer interface in chloroform to different initial surface pressures. The resulting monolayers were allowed to equilibrate for 30 min before addition of the KA1 domain (His6Kcc4p ) to a final concentration of 0.5 μM into the subphase solution. The increase in surface pressure was then monitored for 30 min with constant stirring of the subphase following protein addition, with maximal change in surface pressure typically being reached after 15 min. Lipid mixtures used were: pure stearoyl-oleoyl phosphatidylcholine (SOPC, gray points), SOPC containing 20% (mole/mole) SOPS (red points) or SOPC containing 10% (mole/mole) PtdIns(4,5)P2 (blue points). The increase in surface pressure achieved by addition of Kcc4p-KA1 (vertical axis) is plotted for a series of initial surface pressures (horizontal axis). The critical surface pressure ∏c is the surface pressure above which monolayer penetration is no longer seen. For pure SOPC membranes, this value was 25.5 ± 0.2 mN/m, which is below the estimated cell membrane surface pressure of mN/m (Demel, 1994; Marsh, 1996). Thus, Kcc4p-KA1 insertion into pure PC membranes is unlikely in physiological membranes. However, critical surface pressure values of 30.7 ± 0.3 mN/m and 30.4 ± 0.4 mN/m were measured for SOPS/SOPC and PtdIns(4,5)P2/SOPC monolayers respectively, suggesting that the Kcc4p KA1 domain will insert into membranes with these compositions in vivo. These ∏c values are similar to those previously reported for FYVE, PX, PH, ENTH, and other domains (Cho and Stahelin, 2005; Stahelin et al., 2007).
(B) Analysis of in vitro phospholipid-binding by hMARK1-KA1 variants tested in Figure 6B. SPR binding curves were performed for binding to negatively charged membranes containing 10% (mole/mole) PtdIns(4,5)P2 in a DOPC background. Curves shown are representative of at least two independent experiments. The three mutants that showed impaired PtdIns(4,5)P2 binding also failed to localize to the plasma membrane of yeast cells (Figure 6).
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16. Figure S7
Localization of GFP-Fused Kcc4p Harboring KA1 Domain Mutations, Related to Figure 7
(A) Wild-type or KA1 domain-mutated forms of intact Kcc4p fused to GFP were expressed in wild-type cells or cho1Δ cells (which lack PtdSer). A gallery of 6 representative budding cells is shown for each case, with DIC images at the left and epifluorescence images at the right of each pair. Neither the K1007S/K1010S nor K1016S/K1020S double mutations in the KA1 domain were sufficient to prevent bud neck localization in wild-type cells, whereas the quadruple K1007S/K1010S/K1016S/K1020S mutation abolished GFP/Kcc4p localization to the bud neck. Wild-type GFP/Kcc4p also retains bud neck localization in cho1Δ cells, whereas the reduced plasma membrane charge in these cells was sufficient to cause delocalization of the GFP/Kcc4p variant harboring a K1007S/K1010S double mutation. The behavior seen here represents that of more than 90% of cells imaged in each of three independent experiments (>100 cells each).
(B) Western blotting analysis of yeast cell lysates using a GFP antibody (Covance) illustrates similar expression levels (with no significant free GFP) for wild-type and mutated variants of GFP/Kcc4p. Samples were prepared by boiling ∼10–20 ODs of cells grown to log phase in SDS-PAGE gel-loading buffer for 5–7 min followed by SDS-PAGE and immunoblotting.
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