Supplementary Materials and Methods
Plasmids and cloning
pCDNA3.1myc∆Np63: 2.0 l of cDNA from MCF-7 were used in standard
PCR to amplify ∆Np63 for 30 cycles with the following primers: sense 5’CCCAAGCTTAATACGACTCACTATAGGGAGACCATGGAACAAAAACTCAT
CTCAGAAGAGGATCTGATGTGTACCTGGAAAACAATG-3’, anti-sense 5’CGGGATCCTCACTCCCCCTCCTCTTTG-3’. HindIII and BamHI restriction
sites in sense and antisense primers respectively, are indicated in bold. An Nterminal Myc tag was introduced in each PCR product (sequence highlighted
in 5’ primer). ∆Np63 was cloned into pGEM-T vector system I (Promega),
the HindIII/BamHI fragments containing the complete ∆Np63 cDNA was then
subcloned into the HindIII/BamHI site of pcDNA3 mammalian expression
vector (Invitrogen). Identification of the recombinants was also carried out by
HindIII/BamHI endonuclease digestion with agarose gel electrophoresis and
sequencing.
pCMV-Flag∆Np63: pcDNA3.1∆Np63 was used as template for
amplification of ∆Np63 by PCR with the following primers sense 5’GGATCCAATACGACTCACTATAGGGAGACCATGGAACAAAAACTCATCT
CAGAAGAGGATCTGATGTTGTACCTGGAAAACAATG-3’ and antisense 5’CGGTCGACTCACTCCCCCTCCTCTTTG-3’, BamHI restriction site was
introduced in sense primer and Sal I was introduced into antisense primer
(both in bold), and myc tag was added at the N terminal of p63 (sequence
highlighted in sense primer). PCR products were cloned into pGEM-T vector
system I (Promega). The BamHI/ Sal I fragments containing the complete
∆Np63 cDNAs was then subcloned into BamHI/ Sal I sites of pCMV-Flag
2B vector (invitrogen).
pGEX6p1-∆Np63: pcDNA3.1-∆Np63 was used as template for
amplification of ∆Np63 by PCR with the following primers sense 5’GGATCCATGTTGTACCTGGAAAACAATG-3’ and antisense 5’-CTCGAGTC
ACTCCCCCTCCTCTTTG-3’, BamHI restriction site was introduced in sense
primer and Xho I was introduced into antisense primer ( both in bold). PCR
products were cloned into pGEM-T vector system I (Promega). The BamHI/
Xho I fragments containing the complete ∆Np63 cDNAs was then
subcloned into BamHI/ Xho I sites of pGEX 6p1 vector (GE).
pcDNA3.1-c-Abl constructs:cDNA from K562 cell line was used for cloning
of c-ABL. The following sense primers were used for full length, SH3 and
SH3,SH2 c-Abl constructs: Full length c-ABL (1-1149aa) 5’-GGATCC
GAGACCATGGAACAAAAACTCATCTCAGAAGAGGATCTGATGGGGCAGC
AGCCTGGAAA-3’; SH3 (141-1149aa) 5’-GGATCCGAGACCATGGAAC
AAAAACTCATCTCAGAAGAGGATCTGCTGGAGAAACACTCCTGGTA-3’;
SH3,SH2 5’-GGATCCGAGACCATGGAACAAAAACTCATCTCAGAAGAG
GATCTGGTGGCCGACGGGCTCATCA-3’. Antisense primer for all c-ABL
constructs:5’-CTCGAGCTACCTCTGCACTATGTCAC-3’. BamH I and XhoI
restriction sites were introduced into sense and antisense primers respectively
(both in bold). PCR products were cloned into pGEM-T vector system I
(Promega). The BamHI/ Xho I fragments of c-ABL were then subcloned into
BamHI/ Xho I sites of pcDNA3.1(+) vector (invitrogen).
pEGFP-YAP was described elsewhere (Strano et al., 2001). pCMV-FlagYAP:pEGFP-YAP was used as template for amplification of YAP using 5’GGACGAATTCGATCCCGGGCAGCAG (introducing EcoR1 site) as sense
and 5’-TGGCGTCGACC T ATAACCATGTAAGAAAGC (introducing Sal1 site)
as antisense primers. PCR product was subcloned in to pCMV-Flag 2B.
Mutagenesis
Mutants of Np63 and YAP were constructed using QuickChange Sitedirected Mutagenesis Kit (Stratagene) according to manufacturer’s instruction.
p63 phosphosite tyrosine (Y) to phenylalanine (F) mutants used pCMVflag-2B-∆Np63 as template for the following primers: ∆Np63 (Y55F) sense
5’-GCGCCCTCGCCCTTTGCACAGCCCAGC-3’ and antisense 5’-GCTGGG
CTGTGCAAAGGGCGAGGGCGC-3’; ∆Np63 (Y137F) sense 5'-TCCGCG C
CATGCCTGTCTTCAAAAAAGCTGAGCACGTC-3' and antisense 5'-GAC
GTGCTCAGCTTTTTTGAAGACAGGCATGGCGCGGA-3’; ∆Np63 (Y308F)
sense 5’- CCAGATGATGAACTGTTATTCTTACCAGTGAGGGGCCGTGAG3’ and antisense 5’-CTCACGGCCCCTCACTGGTAAGAATAACAGTTCATC
ATCTGG-3’; Triple phosphosite mutant YYYFFF is made from sequential
mutation from each p63 phosphosite mutant.
pCDNA3.1myc-Np63Y449F(PPXY motif point mutant) used pcDNA3.1
myc ∆Np63 as template for the following primers: sense 5`CACCCCCACCTCCGTTCCCCACAGATTGCAGC–3’ and antisense 5`GCTGCAATCTGTGGGGAACGGAGGTGGGGGTG-3’
Silent p63 phosphosite mutants (Y55F, Y137F and Y308F resistant to
siRNA p63) used corresponding p63 phosphosite mutants as template for
these primers: sense 5’-TGTTCATCATGCCTCGACTATTTCACGACCCAG
GGGC-3’ and antisense 5’-GCCCCTGGGTCGTGAAATAGTCGAGGCA
TGATGAACA-3’
pCMV-Flag YAPY357F (YAP-c-Abl phopshosite mutant) was constructed
with pCMV-Flag YAP as template with these primers: YAP 357F sense 5’GGACTA AGCATGAGCAGCTTTAGTGTCCCTCGAACCCC-3’ and YAP
357F anti-sense 5’-GGGGTTCGAGGGACACTAAAGCTGCTCATGCTTAGT
CC-3’. Letters in primer sequence bolded are sites that were mutated.
Mutations were verified by sequencing.
Supplemental Figure Legends
Figure S1.
Mass spectrometric analysis of in vitro (A) and in vivo (B) c-Abl
phosphorylation of ∆Np63 reveal multiple tyrosine phosphorylation
sites.
(A) Recombinant GST ∆Np63 from c-Abl in vitro kinase assays or (B) FLAG∆Np63 immunoprecipitated from c-Abl co-transfected 293 cells were
fractionated by SDS-PAGE and ∆Np63 band excised, digested by indicated
enzymes and subjected to mass spectrometric analysis as described in
Experimental Procedures. Independent peptides containing phosphorylated
tyrosine residues are listed.
Figure S2.
Rescue mutant constructs of ∆Np63 and ∆Np63YYYFFF are resistant
to p63 siRNA oligonucleotide.
293 cells were reverse transfected with control or p63 siRNA oligonucleotide
and then forward transfected with indicated plasmids. Whole cell lysates were
analyzed with indicated antibodies.