10 research outputs found
SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia
Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML
Intramolecular Cleavage of the hASRGL1 Homodimer Occurs in Two Stages
The
human asparaginase-like protein 1 (hASRGL1) is a member of
the N-terminal nucleophile (Ntn) family that hydrolyzes l-asparagine and isoaspartyl-dipeptides. The nascent protein folds
into an αβ–βα sandwich fold homodimer
that cleaves its own peptide backbone at the G167–T168 bond,
resulting in the active form of the enzyme. However, biophysical studies
of hASRGL1 are difficult because of the curious fact that intramolecular
cleavage of the G167–T168 peptide bond reaches only ≤50%
completion. We capitalized upon our previous observation that intramolecular
processing increases thermostability and developed a differential
scanning fluorimetry assay that allowed direct detection of distinct
processing intermediates for the first time. A kinetic analysis of
these intermediates revealed that cleavage of one subunit of the hASRGL1
subunit drastically reduces the processing rate of the adjacent monomer,
and a mutagenesis study showed that stabilization of the dimer interface
plays a critical role in this process. We also report a comprehensive
analysis of conserved active site residues and delineate their relative
roles in autoprocessing and substrate hydrolysis. In addition to glycine,
which was previously reported to selectively accelerate hASRGL1 cleavage,
we identified several novel small molecule activators that also promote
intramolecular processing. The structure–activity analysis
supports the hypothesis that multiple negatively charged small molecules
interact within the active site of hASRGL1 to act as a base in promoting
cleavage. Overall, our investigation provides a mechanistic understanding
of the maturation process of this Ntn hydrolase family member
The protein phosphatase PPM1A dephosphorylates and activates YAP to govern mammalian intestinal and liver regeneration.
The Hippo-YAP pathway responds to diverse environmental cues to manage tissue homeostasis, organ regeneration, tumorigenesis, and immunity. However, how phosphatase(s) directly target Yes-associated protein (YAP) and determine its physiological activity are still inconclusive. Here, we utilized an unbiased phosphatome screening and identified protein phosphatase magnesium-dependent 1A (PPM1A/PP2Cα) as the bona fide and physiological YAP phosphatase. We found that PPM1A was associated with YAP/TAZ in both the cytoplasm and the nucleus to directly eliminate phospho-S127 on YAP, which conferring YAP the nuclear distribution and transcription potency. Accordingly, genetic ablation or depletion of PPM1A in cells, organoids, and mice elicited an enhanced YAP/TAZ cytoplasmic retention and resulted in the diminished cell proliferation, severe gut regeneration defects in colitis, and impeded liver regeneration upon injury. These regeneration defects in murine model were largely rescued via a genetic large tumor suppressor kinase 1 (LATS1) deficiency or the pharmacological inhibition of Hippo-YAP signaling. Therefore, we identify a physiological phosphatase of YAP/TAZ, describe its critical effects in YAP/TAZ cellular distribution, and demonstrate its physiological roles in mammalian organ regeneration
Mapping the Phosphorylation Pattern of <i>Drosophila melanogaster</i> RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry
Phosphorylation of
the C-terminal domain of RNA polymerase II (CTD)
plays an essential role in eukaryotic transcription by recruiting
transcriptional regulatory factors to the active polymerase. However,
the scarcity of basic residues and repetitive nature of the CTD sequence
impose a huge challenge for site-specific characterization of phosphorylation,
hindering our understanding of this crucial biological process. Herein,
we apply LC-UVPD-MS methods to analyze post-translational modification
along native sequence CTDs. Application of our method to the <i>Drosophila melanogaster</i> CTD reveals the phosphorylation
pattern of this model organism for the first time. The divergent nature
of fly CTD allows us to derive rules defining how flanking residues
affect phosphorylation choice by CTD kinases. Our data support the
use of LC-UVPD-MS to decipher the CTD code and determine rules that
program its function