Target Discovery Global Profiling of Kinases During Hematopoietic Differentiation

Hematopoietic differentiation

  • HL60s are promyeloblastic cells that can be terminally differentiated into either macrophages or granulocytes with phorbol myristate acetate (PMA) and retinoic acid (RA) respectively
  • This differentiation process is an important model for myelocytic leukemia

Goals

The KiNativ™ platform was used to profile changes in activity/abundance of all protein kinases during this differentiation process:

  • To better understand the molecular mechanism of differentiation
  • To identify novel targets for the treatment of myelocytic leukemia

Profiling kinases in HL60 cells during PMA and RA differentiation

  • The protein and lipid kinases in resting, PMA, and RA differentiated HL60 cells were analyzed by targeted mass spectrometry.
  • A total of 194 kinase peptides comprising 175 unique protein and lipid kinases were accurately quantified

Distribution of probe labeling changes (number of kinases)

Summary of Results from KiNativ™ Profiling

Changes in probe labeling for selected functional classes of kinases

MS2 Ion Traces for Selected Peptides

Changes in activity versus abundance

To determine if the changes in probe labeling were due to changes in activity or abundance, the abundance of selected kinases was directly determined by Western blot. Changes in PKAC, IKKb, and CDK6 probe labeling did not correlate with changes in abundance.

Comparisons of MS Results with Western Blots

The mechanism for reduced probe labeling of CDK6 during differentiation was determined by monitoring the abundance of CDK6 regulators.

  • CDK6 probe labeling decreased 50- and 10-fold during PMA and RA differentiation, respectively
  • By Western blot, CDK6 only decreased 2-fold during PMA differentiation, and was unchanged during RA differentiation
  • The strong decrease in probe labeling of CDK6 correlated with increases in the known CDK inhibitors p27 (PMA and RA) and p21 (PMA)

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