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Formyl-Met-Leu-Phe-dependent serine kinase for a 64,000 molecular weight protein of polymorphonuclear leukocytes in a
1Department of Antibiotics, National Institute of Health, Tokyo, Japan.
Abstract:
In the Triton X-100-treated polymorphonuclear leukocytes (PMN), which were stimulated with formyl-Met-Leu-Phe (FMLP) for 1 min, a 64,000 molecular weight protein (p64) was preferentially phosphorylated by the incubation with [gamma-32P]ATP in the presence of Mg2+, but not in the presence of Ca2+. Phosphoamino acid analysis of pp64 revealed that the p64-kinase was a serine-specific protein kinase. The p64 was maximally phosphorylated in the first minute, suggesting that the rapid phosphorylation was related to the initial reaction for activation of the FMLP-stimulated PMN functions. The FMLP-stimulated phosphorylation of p64 was slightly inhibited by the addition of cGMP in the reaction mixture. However, addition of cAMP, the cyclic nucleotide-dependent kinase inhibitor (H-8), protein kinase C-inhibitor (H-7) or Ca/calmodulin-dependent kinase inhibitor (W-7), showed no effect on the phosphorylation. These data suggest that phosphorylation of p64 seems to be a novel protein kinase specific to p64.
Insights
Researchers identified a novel serine-specific protein kinase that rapidly phosphorylates a 64 kDa protein (p64) in polymorphonuclear leukocytes (PMN) upon stimulation. This phosphorylation is crucial for initial cell activation.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Polymorphonuclear leukocytes (PMN) are critical immune cells involved in inflammatory responses.
- Formyl-Met-Leu-Phe (FMLP) is a potent chemoattractant that activates PMNs.
- Protein phosphorylation plays a key role in cellular signaling and activation.
Purpose of the Study:
- To investigate the rapid protein phosphorylation events in FMLP-stimulated PMNs.
- To identify novel kinases involved in PMN activation.
Main Methods:
- Triton X-100 treated PMNs were stimulated with FMLP.
- Phosphorylation was assessed using [gamma-32P]ATP and Mg2+.
- Phosphoamino acid analysis was performed on the phosphorylated protein.
- The effect of various inhibitors and cyclic nucleotides was evaluated.
Main Results:
- A 64,000 molecular weight protein (p64) was rapidly and preferentially phosphorylated in FMLP-stimulated PMNs.
- The phosphorylation occurred in the presence of Mg2+ but not Ca2+.
- Phosphoamino acid analysis identified the kinase as serine-specific.
- Cyclic guanosine monophosphate (cGMP) slightly inhibited phosphorylation, while other inhibitors had no effect.
Conclusions:
- The data suggest the existence of a novel, serine-specific protein kinase responsible for p64 phosphorylation.
- This rapid phosphorylation event is likely involved in the initial activation of FMLP-stimulated PMN functions.