2. Protein Interaction Partners of Protein Phosphatase 2A Catalytic Subunit
in Rat β-Islet cells Using Quantitative Mass Spectrometry
Divyasri Damacharla1,2; Khadija Syeda1,2 Xiangmin Zhang2; Danjun Ma2; Yue Qi2; Anjaneyulu Kowluru1,2; Zhengping Yi2
1John D. Dingell VA Medical Center and 2Wayne State University, Detroit, MI
INTRODUCTION
• Protein phosphatase 2A (PP2A) is one of the major
serine/threonine phosphatases
• Its activity is up regulated under glucolipotoxic
conditions in liver, retina and pancreatic islet cells
However, it remains unknown about the various
interactions of PP2Ac in rat pancreatic β islet cells that
may contribute to the increased PP2A activity and
regulation of cellular function
• Here, using HPLCE-ESI-MS/MS, we aimed to identify
the PP2Ac interaction partners involved in insulin
secretion or other pathways which may lead to
dysfunction or demise of the β-islet cells
METHODS
INS-1 832/13 cells
LOW GLUCOSE HIGH GLUCOSE
IMMUNOPRECIPITATION
SDS-PAGE
IN-GEL TRYPSIN DIGESTION; PEPTIDE ENRICHMENT
HPLC-MS/MS
MAXQUANT AND STATISTICALANALYSIS
RESULTS
SUMMARY
Proteins identified with minimum 2 unique peptides with FDR at
0.01 in at least one PP2Ac IP? (1131 proteins)
Identified with LFQ peak area (PA) in more than half (e.g., > 4
out of 8) PP2Ac IP samples? (514 proteins,)
Enrichment ratio for each protein determined. Enrichment ratio for
a protein > 10 (PP2Ac vs NIgG IP)? (606 proteins)
Normalized PA for PP2Ac interaction partners determined
P < 0.05 by independent t tests?
(89 proteins)
With a fold change greater than 1.5 (i.e., 1.5 fold increase) or
less than 0.67 (i.e., 1.5 fold decrease) HG vs. LG? (265 proteins)
Figure 1 statistical analysis and summary of the results obtained
• Largest PP2A interaction network till date in rat β-
islet cells
• Validation of the identified partners will give insight
into the regulation of PP2A
• Further validation of glucose responsive partners
will help understand role of PP2A under high
glucose conditions
• Significant complexes may be targeted later to alter
the composition and thereby prevent the demise of
the β-islet cells under glucotoxic conditions
• 514 PP2Ac interaction
partners
• 38 previously reported
leaving 476 novel partners
• 89 showed significant
difference in response to high
glucose treatment
• Ingenuity Pathway Analysis
of the 514 partners revealed
59 significantly enriched
pathways
• Manual literature search
revealed a number of
proteins involved in insulin
secretion and other related
cell functions (Fig 2)
Protein synthesis
RPL4, PRL9, RPL18A,
RPL30, EIF2B1
Vesicle trafficking
VPS52, VPS37A,
TSG101, Rab10, Eea1,
Rab5C
Protein degradation
UBR1, UBE2C, CNOT4,
CBR3
Membrane fusion
VAT1, SLMAP, Rab5c
Gluconeogenesis
WDR5
Anti-apoptosis
PPP4R1, CIAPIN1
Transcription control
VGLL4, TRIP11, STAT6,
PHF5A , NcoR1,
HIST1H1C, DDX17
PTM
UGGT1, WDR5, LIMK1, PPM1B,
PPP2R1B, PPP2R2A, PPP4R1, PPP6c
Membrane reshaping
TXNRD1, SLMAP,
SH3BP1
Insulin secretion
RhoA, Rab5c, PLA2G6,
PFKFB2, EIF2C2, APPL1
DNA damage/repair
LIG1, INST5, INST7,
INST12
Protein sorting, trafficking
GGA2, SRP72
Glucose flux control
GFPT1
PP2Ac
Figure 2 partners involved in various important functions
RESULTS
