2 Material and methods
3.1 RNAi screen for PIP-metabolizing enzymes in neuronal membrane trafficking
3.2.1 PPK-2 is orthologous to mammalian PI5P 4-kinases
The following section will focus on the comparison of C. elegans PPK-2 to its mammalian orthologs.
Phylogenetic distances of PPK-2 to its known mammalian orthologs have been determined and are illustrated in Figure 11. Since the mammalian Type II PIP kinases are highly conserved to each other, enzymes of the same isoform of different organisms show similar phylogenetic distances to PPK-2. In summary, all respective Type II PIP kinase isoforms show comparably high homology to C. elegans PPK-2, owing between 47 and at most 52 % conserved amino acids (Appendix). For comparison sake, a sequence alignment with PPK-2 orthologs identified in other model organisms such as zebra fish and fruit fly can be found in the Appendix.
55 Figure 11 Phylogenetic tree of C. elegans PPK-2 and mammalian orthologs
Enzymes of the same isoform of different organisms show similar phylogenetic distances to PPK-2. H. s., H. sapiens; M. m., M. musculus; R. n., R. norvegicus; scale bar, 0.1 substitutions per amino acid site.
The structurally best characterized mammalian Type II PIP kinase is the beta isoform of H. sapiens whose structure was solved by crystallization. Beside this structural information also catalytically important amino acids have been identified. Thus, substantiated information linking primary sequence information with tertiary structure-dependent function is available for this protein (Rao et al., 1998; Kunz et al., 2000; Kunz et al., 2002).
In order to assign a putative function to amino acids in the primary sequence of PPK-2, a direct comparison to H. sapiens Type II PIP kinase beta has been carried out. To compare the C. elegans kinase to all three mammalian Type II PIP kinase isoforms, the two remaining human isoforms alpha and gamma have been considered for the sequence alignment as well.
The multiple sequence alignment shown in Figure 12 demonstrates the high conservation between PPK-2 and any of the human Type II PIP kinase isoforms. Beside the overall sequence similarity also essential amino acids for catalysis and membrane interaction predicted for H. sapiens Type II PIP kinase beta are conserved (Rao et al., 1998).
Most importantly, the so-called activation loop of Type II PIP kinases is found to be conserved in the C-terminus of PPK-2. The activation loop was demonstrated to be crucial for subcellular localization (Kunz et al., 2000) and furthermore one of its comprising amino acids determines substrate specificity for PI5P (Kunz et al., 2002). This particular alanine is conserved throughout all Type II PIP kinases, including PPK-2.
Since the primary sequence of PPK-2 is very similar to its human orthologs, it is likely that also the secondary and tertiary structure is conserved. In order to test this, a modeling attempt for PPK-2 has been done. Indeed, it was possible to obtain a tertiary structure model of PPK-2 when using the structure of H. sapiens Type II PIP kinase beta as a template. To prelude this, a brief summary PPK-2 M. m. PIP4KIIgamma
R. n. PIP4KIIgamma H. s. PIP4KIIgamma M. m. PIP4KIIbeta
H. s. PIP4KIIbeta R. n. PIP4KIIbeta M. m. PIP4KIIalpha
H. s. PIP4KIIalpha
R. n. PIP4KIIalpha
0.1
Type II alpha isoforms
Type II beta isoforms
Type II gamma isoforms
56 addressing the structure and some characteristics of H. sapiens Type II PIP kinase beta is given in the following section, leading to the template based modeling of PPK-2.
PIP4KIIalpha 1 ---MATPGNLGSSVLAS---KTKTKKKHFVAQKVKLFRASDPLLSVLMWGVNHSIN PIP4KIIbeta 1 MSSNCTSTTAVAVAPLSAS---KTKTKKKHFVCQKVKLFRASEPILSVLMWGVNHTIN PIP4KIIgamma 1 MASSSVPPATVSAATAGPGPGFGFASKTKKKHFVQQKVKVFRAADPLVGVFLWGVAHSIN PPK-2 1 ---MSTKKKTKVLSK---KKGKILVPKWKLFRAKEPVLSVFMWGINHTVD
consensus 1 . . ... ... .. ....**.... .*.*.***..*...* .**..*...
+ + + + + + +
PIP4KIIalpha 51 ELSHVQIPVMLMPDDFKAYSKIKVDNHLFNKENMPSHFKFKEYCPMVFRNLRERFGIDDQ PIP4KIIbeta 56 ELSNVPVPVMLMPDDFKAYSKIKVDNHLFNKENLPSRFKFKEYCPMVFRNLRERFGIDDQ PIP4KIIgamma 61 ELSQVPPPVMLLPDDFKASSKIKVNNHLFHRENLPSHFKFKEYCPQVFRNLRDRFGIDDQ PPK-2 45 QLLHVPPPGLLMPDDFKAYSKVKIDNHNFNKDIMPSHYKVKEYCPNVFRNLREQFGVDNF consensus 61 .*..*..*..*.******.**.*..**.*...**..*.*****.******..**.*..
*# * * * * + ++
PIP4KIIalpha 111 DFQNSLTRSAPLP---NDSQARSGARFHTSYDKRYIIKTITSEDVAEMHNILKKYHQYIV PIP4KIIbeta 116 DYQNSVTRSAPIN---SDSQGRCGTRFLTTYDRRFVIKTVSSEDVAEMHNILKKYHQFIV PIP4KIIgamma 121 DYLVSLTRNPPSE---SEG---SDGRFLISYDRTLVIKEVSSEDIADMHSNLSNYHQYIV PPK-2 105 EYLRSLTSYEPEPDLLDGSAKDSTPRFFISYDKKFVIKSMDSEAVAELHSVLRNYHQYVV consensus 121 .. .*.*...*.. ...**. .**....**...**..*..* .*. ***..*
+ * *** # #
PIP4KIIalpha 168 ECHGITLLPQFLGMYRLNVDGVEIYVIVTRNVFSHRLSVYRKYDLKGSTVAREASDKEKA PIP4KIIbeta 173 ECHGNTLLPQFLGMYRLTVDGVETYMVVTRNVFSHRLTVHRKYDLKGSTVAREASDKEKA PIP4KIIgamma 175 KCHGNTLLPQFLGMYRVSVDNEDSYMLVMRNMFSHRLPVHRKYDLKGSLVSREASDKEKV PPK-2 165 EKQGKTLLPQYLGLYRLTIEGSETYLIVMRNVFGRKYGVHTKFDLKGSTVSRAASDKEKA consensus 181 ...*.*****.**.**...*..* **.*...*..*.*****.* *.******. PPK-2 225 KDLPTLKDNDFLEQNWKLNLPPEAGKLLIEMLTSDTEWLTRMHLMDYSLLVGIHDCERAA consensus 241 *.***.**.**... .*... .*...*.*..*.*.*...******.****..*..
PIP4KIIalpha 288 QEEVECEENDGEEEGESDGT---HPVGTPPDSPGNTLNSSPPLAPGEFDPNIDVY PIP4KIIbeta 293 QEEMEVEERAEDEECENDGVGG---NLLCSYGTPPDSPGNLLSFPRFFGPGEFDPSVDVY PIP4KIIgamma 295 EPEEEAPVREDESEVDGDCSLTGPPALVGSYGTSPEGIGGYIHSHRPLGPGEFESFIDVY PPK-2 285 QEAANRPVEQNSEESGDELAP---TPPDSPIPSTGGAFPGVSGGPDLDDEFY consensus 301 ... ...* . ... .. ...*.*... . ...*.... ...*
** #
PIP4KIIalpha 340 GIKCHENSPRKEVYFMAIIDILTHYDAKKKAAHAAKTVKHGAGAE-ISTVNPEQYSKRFL PIP4KIIbeta 350 AMKSHESSPKKEVYFMAIIDILTPYDTKKKAAHAAKTVKHGAGAE-ISTVNPEQYSKRFN PIP4KIIgamma 355 AIRSAEGAPQKEVYFMGLIDILTQYDAKKKAAHAAKTVKHGAGAE-ISTVHPEQYAKRFL PPK-2 334 AIASPADFEKNLIYFIGLVDILTYYGIKKRSATAAKTVKYGSDAENISTVKPEQYAKRLV consensus 361 ... ...**....**** *..**..*.******.*..** ****.**** **..
Figure 12 Primary sequence alignment of PPK-2 and human orthologs
The primary sequence of PPK-2 was aligned with all three PI5P 4-kinase isoforms of H. sapiens (PIP4KIIalpha, PIP4KIIbeta, PIP4KIIgamma). Conserved and similar amino acids are shaded in black and grey, respectively. The variable insert is colored in blue and the activation loop is highlighted in red letters with the conserved alanin essential for substrate specificity (Kunz et al., 2000; Kunz et al., 2002) shown in yellow. Functional amino acids in ATP binding (*), membrane binding (+), and PI5P binding (#) are marked according to Rao et al., 1998.
The tertiary structure of H. sapiens
domain. Both domains in turn are built of an antiparallel beta sheet form mainly the exterior surface of the protein (Figu
kinase beta is related to the tertiary structure 1999).
The secondary structure of H. sapiens
beta strands. In Figure 13, the Type II PIP kinase beta topology was projected on its primary sequence aligned with the primary sequence of PPK
of Type II PIP kinase beta were found to be mostly 2 hence leading to the conclusion that PPK
Figure 13 Putative secondary structure of PPK Primary sequence alignment of PPK
according secondary structure comprised of alpha helices (spirals) and beta strands (arrows)
et al., 1998). Identical amino acids are shaded in black, well conserved amino acids are shown in bold and are boxed.
H. sapiens Type II PIP kinase beta consists of an N-terminal and a C
domain. Both domains in turn are built of an antiparallel beta sheet, whereas according alpha helices rior surface of the protein (Figure 14A). The folding of H. sapiens
tertiary structure of protein kinases (Rao et al., 1998
H. sapiens Type II PIP kinase beta is comprised of alpha helices as wel , the Type II PIP kinase beta topology was projected on its primary sequence aligned with the primary sequence of PPK-2. Amino acids assigned to secondary structure elements of Type II PIP kinase beta were found to be mostly identical to the corresponding amino acids of PPK 2 hence leading to the conclusion that PPK-2 possesses a similar topology.
Putative secondary structure of PPK-2
Primary sequence alignment of PPK-2 and human Type II PIP kinase beta (PDB ID 1BO1) combined with the comprised of alpha helices (spirals) and beta strands (arrows)
). Identical amino acids are shaded in black, well conserved amino acids are shown in bold and are
57 terminal and a C- terminal whereas according alpha helices H. sapiens Type II PIP ., 1998; Grishin et al.,
is comprised of alpha helices as well as , the Type II PIP kinase beta topology was projected on its primary sequence 2. Amino acids assigned to secondary structure elements identical to the corresponding amino acids of
PPK-D 1BO1) combined with the comprised of alpha helices (spirals) and beta strands (arrows) (according to Rao ). Identical amino acids are shaded in black, well conserved amino acids are shown in bold and are
The crystal structure of H. sapiens regions: the activation loop and the so C-terminal activation loop is essential hence catalytic activity (Kunz et al
loop and highly divergent between all Type II PIP
The H. sapiens Type II PIP kinase beta can form a homodimer (Figure 1 of both monomers. In addition, the N
thought to be important for the lipids (Rao et al., 1998).
Tertiary structure modeling of PPK
kinase beta (PDB ID 1BO1) as a template. As illustrated in Figures 1 indeed very similar to the human enzyme.
This tertiary structure information, combined with the primary sequence alignment and putative secondary structure elements
kinase.
A
C
Figure 14 Modeling of the putative tertiary structure of PPK Ribbon drawings. A, tertiary structure of human Type II
flexible regions of the insert and the activation loop are sketched. B, postulated homodimer of human Type II PIP kinase beta (Rao et al., 1998). C, tertiary struc
monomer A of human Type II PIP kinase beta. D, superposition of A and C.
H. sapiens Type II PIP kinase beta possesses two structural disordered regions: the activation loop and the so-called insert (Figures 12 and 14A). As already mentioned,
terminal activation loop is essential for cellular localization as well as for substrate recognition and et al., 2000). The insert is a region located proximally
loop and highly divergent between all Type II PIP kinases (Heck et al., 2007).
I PIP kinase beta can form a homodimer (Figure 14B), mediated
of both monomers. In addition, the N-terminus contains many basic amino acids and is therefore the interaction with the positively charged head group
Tertiary structure modeling of PPK-2 was possible using the tertiary structure of H. sapiens
kinase beta (PDB ID 1BO1) as a template. As illustrated in Figures 14C and D, its putative folding is ery similar to the human enzyme.
information, combined with the primary sequence alignment and putative secondary structure elements, allows the conclusion that PPK-2 is a bona fide
B
C D
Modeling of the putative tertiary structure of PPK-2
structure of human Type II PIP kinase beta (chain A, PDB ID 1BO1). The highly flexible regions of the insert and the activation loop are sketched. B, postulated homodimer of human Type II ). C, tertiary structure of PPK-2 modeled based on the crystal structure of monomer A of human Type II PIP kinase beta. D, superposition of A and C.
58 terminus contains many basic amino acids and is therefore interaction with the positively charged head groups of membrane
H. sapiens Type II PIP C and D, its putative folding is
information, combined with the primary sequence alignment and derived bona fide Type II PIP
B
(chain A, PDB ID 1BO1). The highly flexible regions of the insert and the activation loop are sketched. B, postulated homodimer of human Type II 2 modeled based on the crystal structure of
59